Compounds and conjugates thereof

ABSTRACT

A conjugate comprising the following topoisomerase inhibitor derivative (A*): with a linker for connecting to a Ligand Unit, wherein the linker is attached in a cleavable manner to the amino residue. The Ligand Unit is preferably an antibody. Also provided is A* with the linking unit attached, and intermediates for their synthesis, as well as the released warhead.

The present invention relates to targeted conjugates comprising aspecific topoisomerase inhibitor and compounds useful in theirsynthesis, as well as the released warhead.

BACKGROUND TO THE INVENTION

Topoisomerase Inhibitors

Topoisomerase inhibitors are chemical compounds that block the action oftopoisomerase (topoisomerase I and II), which is a type of enzyme thatcontrols the changes in DNA structure by catalyzing the breaking andrejoining of the phosphodiester backbone of DNA strands during thenormal cell cycle.

The following compound:

in racemic form was disclosed in EP 0296597 (Example 63). It is alsodisclosed (as compound 34 in racemic form) in Sugimori, M., et al., JMed Chem, 1998, 41, 2308-2318 (DOI: 10.1021/jm970765q), where itsbiological activity is discussed, alongside that of a number of relatedcompounds.

Various topoisomerase inhibitors, such as irinotecan and exatecanderivatives and doxorubicin, have been included in antibody drugconjugates. For example, Daiichi Sankyo have DS-8201a in clinicaltrials:

where the antibody is Her2 (Takegawa, N., et al., Int J Cancer, 2017,141, 1682-1689 (DOI: 10.1002/ijc.30870). This ADC releases the exatecanderivative:

Burke, P. J., et al., Bioconjugate Chem., 2009, 20, 1242-1250, disclosesconjugates of:

which are linked via the amino group with the following structures:

which include a PABC (para-aminobenzyloxycarbonyl) group.

Immunomedics have Sacituzumab Govitecan (IMMU-132) in clinical trials(Cardillo, T. M., et al., Bioconjugate Chem, 2015, 26(5), 919-931, DOI:10.1021/acs.bioconjchem.5b00223)

SUMMARY OF THE INVENTION

In a general aspect the present invention provides a conjugatecomprising the following topoisomerase inhibitor derivative (A*, theDrug Unit):

with a linker for connecting to a Ligand Unit, wherein the linker isattached in a cleavable manner to the amino residue. The Ligand Unit ispreferably an antibody. The invention also provides A* with the linkingunit attached, and intermediates for their synthesis, as well as thereleased warhead.

A first aspect of the present invention comprises a compound with theformula I:

and salts and solvates thereof, wherein R^(L) is a linker for connectionto a Ligand Unit, which is selected from:

-   -   (ia):

-   -   wherein    -   Q is:

where Q^(X) is such that Q is an amino-acid residue, a dipeptideresidue, a tripeptide residue or a tetrapeptide residue;

-   -   X is:

-   -   where a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1,        d=0 to 5, wherein at least b1 or b2=0 (i.e. only one of b1 and        b2 may not be 0) and at least c1 or c2=0 (i.e. only one of c1        and c2 may not be 0);    -   G^(L) is a linker for connecting to a Ligand Unit;    -   (ib):

-   -   where R^(L1) and R^(L2) are independently selected from H and        methyl, or together with the carbon atom to which they are bound        form a cyclopropylene or cyclobutylene group; and    -   e is 0 or 1.

A second aspect of the present invention provides a method of making acompound of the first aspect of the invention, comprising at least oneof the method steps set out below.

In a third aspect, the present invention provides a conjugates offormula IV:

L-(D^(L))_(p)  (IV)

or a pharmaceutically acceptable salt or solvate thereof, wherein L is aLigand unit (i.e., a targeting agent), D^(L) is a Drug Linker unit thatis of formula III:

R^(LL) is a linker connected to the Ligand unit selected from

(ia′):

where Q and X are as defined in the first aspect and G^(LL) is a linkerconnected to a Ligand Unit; and

(ib′):

where R^(L1) and R^(L2) are as defined in the first aspect; and

p is an integer of from 1 to 20.

Accordingly, the Conjugates comprise a Ligand unit covalently linked toat least one Drug unit (A*) by a Linker unit (i.e. a Ligand unit withone or more Drug-Linker units attached). The Ligand unit, described morefully below, is a targeting agent that binds to a target moiety. TheLigand unit can, for example, specifically bind to a cell component (aCell Binding Agent) or to other target molecules of interest.Accordingly, the present invention also provides methods for thetreatment of, for example, various cancers and autoimmune disease. Thesemethods encompass the use of the Conjugates wherein the Ligand unit is atargeting agent that specifically binds to a target molecule. The Ligandunit can be, for example, a protein, polypeptide or peptide, such as anantibody, an antigen-binding fragment of an antibody, or other bindingagent, such as an Fc fusion protein.

The drug loading is represented by p, the number of drug units perLigand unit (e.g., an antibody). Drug loading may range from 1 to 20Drug units (D) per Ligand unit (e.g., Ab or mAb). For compositions, prepresents the average drug loading of the Conjugates in thecomposition, and p ranges from 1 to 20.

A fourth aspect of the present invention provides the use of a conjugateof the third aspect of the invention in the manufacture of a medicamentfor treating a proliferative disease. The fourth aspect also provides aconjugate of the third aspect of the invention for use in the treatmentof a proliferative disease.

One of ordinary skill in the art is readily able to determine whether ornot a candidate compound treats a proliferative condition for anyparticular cell type. For example, assays which may conveniently be usedto assess the activity offered by a particular compound are described inthe examples below.

In Nakada, et al., Bioorg Med Chem Lett, 26 (2016), 1542-1545 (DOI:10.1016/j.bmcl.2016.02.020) discusses a series of ADCs:

and concludes that the decreased cytotoxicity of ADCs (1) and (2) may bedue to the steric hinderance of the released drug moiety on the siteacted on by the degrading enzymes in tumour cells. This document teachesthe importance of spacing the peptidic group from the bulky releaseddrug moiety. In contrast, in the present invention, the peptidic groupis linked directly to the bulky released drug moiety.

A fifth aspect of the present invention is the compound A:

as a single enantiomer or in an enantiomerically enriched form.

A sixth aspect of the present invention is a compound with the formulaVI:

where Q is as defined in the first aspect.

Definitions

C₅₋₆ arylene: The term “C₅₋₆ arylene”, as used herein, pertains to adivalent moiety obtained by removing two hydrogen atoms from an aromaticring atom of an aromatic compound.

In this context, the prefixes (e.g. C₅₋₆) denote the number of ringatoms, or range of number of ring atoms, whether carbon atoms orheteroatoms.

The ring atoms may be all carbon atoms, as in “carboarylene groups”, inwhich case the group is phenylene (C₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in“heteroarylene groups”. Examples of heteroarylene groups include, butare not limited to, those derived from:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₆);

O₁: furan (oxole) (C₅);

S₁: thiophene (thiole) (C₅);

N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂O₁: oxadiazole (furazan) (C₅);

N₃O₁: oxatriazole (C₅);

N₁S₁: thiazole (C₅), isothiazole (C₅);

N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅),pyridazine (1,2-diazine) (C₆), pyrimidine (1,3-diazine) (C₆) (e.g.,cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆); and

N₃: triazole (C₅), triazine (C₆).

C₁₋₄ alkyl: The term “C₁₋₄ alkyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a carbonatom of a hydrocarbon compound having from 1 to 4 carbon atoms, whichmay be aliphatic or alicyclic, and which may be saturated or unsaturated(e.g. partially unsaturated, fully unsaturated). The term “C_(1-n)alkyl” as used herein, pertains to a monovalent moiety obtained byremoving a hydrogen atom from a carbon atom of a hydrocarbon compoundhaving from 1 to n carbon atoms, which may be aliphatic or alicyclic,and which may be saturated or unsaturated (e.g. partially unsaturated,fully unsaturated). Thus, the term “alkyl” includes the sub-classesalkenyl, alkynyl, cycloalkyl, etc., discussed below.

Examples of saturated alkyl groups include, but are not limited to,methyl (CA ethyl (C2), propyl (C₃) and butyl (C₄).

Examples of saturated linear alkyl groups include, but are not limitedto, methyl (C₁), ethyl (C₂), n-propyl (C₃) and n-butyl (C₄).

Examples of saturated branched alkyl groups include iso-propyl (C₃),iso-butyl (C₄), sec-butyl (C₄) and tert-butyl (C₄).

C₂₋₄ Alkenyl: The term “C₂₋₄ alkenyl” as used herein, pertains to analkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to,ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃), 2-propenyl (allyl,—CH—CH═CH₂), isopropenyl (1-methylvinyl, —C(CH₃)═CH₂) and butenyl (C₄).

C₂₋₄ alkynyl: The term “C₂₋₄ alkynyl” as used herein, pertains to analkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to,ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH₂—C≡CH).

C₃₋₄ cycloalkyl: The term “C₃₋₄ cycloalkyl” as used herein, pertains toan alkyl group which is also a cyclyl group; that is, a monovalentmoiety obtained by removing a hydrogen atom from an alicyclic ring atomof a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, thosederived from:

-   -   saturated monocyclic hydrocarbon compounds:

cyclopropane (C₃) and cyclobutane (C₄); and

-   -   unsaturated monocyclic hydrocarbon compounds:

cyclopropene (C₃) and cyclobutene (C₄).

Connection labels: In the formula

the superscripted labels ^(C(═O)) and ^(NH) indicate the group to whichthe atoms are bound. For example, the NH group is shown as being boundto a carbonyl (which is not part of the moiety illustrated), and thecarbonyl is shown as being bound to a NH group (which is not part of themoiety illustrated).

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66,1-19 (1977).

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g. —COOH may be —COO⁻), then a salt may be formed witha suitable cation. Examples of suitable inorganic cations include, butare not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earthcations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³. Examplesof suitable organic cations include, but are not limited to, ammoniumion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺,NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions arethose derived from: ethylamine, diethylamine, dicyclohexylamine,triethylamine, butylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline,meglumine, and tromethamine, as well as amino acids, such as lysine andarginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g. —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, trifluoroacetic acidand valeric. Examples of suitable polymeric organic anions include, butare not limited to, those derived from the following polymeric acids:tannic acid, carboxymethyl cellulose.

Solvates

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

Isomers

Certain compounds of the invention may exist in one or more particulargeometric, optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R—, S—, and meso-forms; D- and L-forms; d-and l-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand l or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or l meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

“Enantiomerically enriched form” refers to a sample of a chiralsubstance whose enantiomeric ratio is greater than 50:50 but less than100:0.

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g. C₁-7 alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/enediamine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Examples of isotopes that can be incorporated into compounds of theinvention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, chlorine and iodine, such as, but not limited to²H (deuterium, D), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F, ³¹P, ³²P, ³⁵S,³⁶Cl, and ¹²⁵I. Various isotopically labeled compounds of the presentinvention, for example those into which radioactive isotopes such as 3H,13C, and 14C are incorporated. Such isotopically labelled compounds maybe useful in metabolic studies, reaction kinetic studies, detection orimaging techniques, such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) including drug orsubstrate tissue distribution assays, or in radioactive treatment ofpatients. Deuterium labelled or substituted therapeutic compounds of theinvention may have improved DMPK (drug metabolism and pharmacokinetics)properties, relating to distribution, metabolism, and excretion (ADME).Substitution with heavier isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements. An18F labeled compound may be useful for PET or SPECT studies.Isotopically labeled compounds of this invention and prodrugs thereofcan generally be prepared by carrying out the procedures disclosed inthe schemes or in the examples and preparations described below bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent. Further, substitution with heavierisotopes, particularly deuterium (i.e., 2H or D) may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements or animprovement in therapeutic index. It is understood that deuterium inthis context is regarded as a substituent. The concentration of such aheavier isotope, specifically deuterium, may be defined by an isotopicenrichment factor. In the compounds of this invention any atom notspecifically designated as a particular isotope is meant to representany stable isotope of that atom.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.asymmetric synthesis) and separation (e.g. fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

Ligand Unit

The Ligand Unit may be of any kind, and include a protein, polypeptide,peptide and a non-peptidic agent that specifically binds to a targetmolecule. In some embodiments, the Ligand unit may be a protein,polypeptide or peptide. In some embodiments, the Ligand unit may be acyclic polypeptide. These Ligand units can include antibodies or afragment of an antibody that contains at least one targetmolecule-binding site, lymphokines, hormones, growth factors, or anyother cell binding molecule or substance that can specifically bind to atarget.

The terms “specifically binds” and “specific binding” refer to thebinding of an antibody or other protein, polypeptide or peptide to apredetermined molecule (e.g., an antigen).

Typically, the antibody or other molecule binds with an affinity of atleast about 1×10⁷ M⁻¹, and binds to the predetermined molecule with anaffinity that is at least two-fold greater than its affinity for bindingto a non-specific molecule (e.g., BSA, casein) other than thepredetermined molecule or a closely-related molecule.

Examples of Ligand units include those agents described for use in WO2007/085930, which is incorporated herein.

In some embodiments, the Ligand unit is a Cell Binding Agent that bindsto an extracellular target on a cell. Such a Cell Binding Agent can be aprotein, polypeptide, peptide or a non-peptidic agent. In someembodiments, the Cell Binding Agent may be a protein, polypeptide orpeptide. In some embodiments, the Cell Binding Agent may be a cyclicpolypeptide. The Cell Binding Agent also may be antibody or anantigen-binding fragment of an antibody. Thus, in one embodiment, thepresent invention provides an antibody-drug conjugate (ADC).

Cell Binding Agent

A cell binding agent may be of any kind, and include peptides andnon-peptides. These can include antibodies or a fragment of an antibodythat contains at least one binding site, lymphokines, hormones, hormonemimetics, vitamins, growth factors, nutrient-transport molecules, or anyother cell binding molecule or substance.

Peptides

In one embodiment, the cell binding agent is a linear or cyclic peptidecomprising 4-30, preferably 6-20, contiguous amino acid residues.

In one embodiment the cell binding agent comprises a peptide that bindsintegrin α_(v)β₆. The peptide may be selective for α_(v)β₆ over XYS.

In one embodiment the cell binding agent comprises the A20FMDV-Cyspolypeptide. The A20FMDV-Cys has the sequence: NAVPNLRGDLQVLAQKVARTC.Alternatively, a variant of the A20FMDV-Cys sequence may be used whereinone, two, three, four, five, six, seven, eight, nine or ten amino acidresidues are substituted with another amino acid residue. Furthermore,the polypeptide may have the sequence NAVXXXXXXXXXXXXXXXRTC.

Antibodies

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), multivalent antibodies and antibody fragments, so long asthey exhibit the desired biological activity (Miller et al (2003) Jour.of Immunology 170:4854-4861). Antibodies may be murine, human,humanized, chimeric, or derived from other species. An antibody is aprotein generated by the immune system that is capable of recognizingand binding to a specific antigen. (Janeway, C., Travers, P., Walport,M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, N.Y.).A target antigen generally has numerous binding sites, also calledepitopes, recognized by CDRs on multiple antibodies. Each antibody thatspecifically binds to a different epitope has a different structure.Thus, one antigen may have more than one corresponding antibody. Anantibody includes a full-length immunoglobulin molecule or animmunologically active portion of a full-length immunoglobulin molecule,i.e., a molecule that contains an antigen binding site thatimmunospecifically binds an antigen of a target of interest or partthereof, such targets including but not limited to, cancer cell or cellsthat produce autoimmune antibodies associated with an autoimmunedisease. The immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD,and IgA), class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclassof immunoglobulin molecule. The immunoglobulins can be derived from anyspecies, including human, murine, or rabbit origin.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and scFv fragments;diabodies; linear antibodies; fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, CDR (complementarydetermining region), and epitope-binding fragments of any of the abovewhich immunospecifically bind to cancer cell antigens, viral antigens ormicrobial antigens, single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature 256:495, or may be made byrecombinant DNA methods (see, U.S. Pat. No. 4,816,567). The monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in Clackson et al (1991) Nature, 352:624-628; Markset al (1991) J. Mol. Biol., 222:581-597 or from transgenic mice carryinga fully human immunoglobulin system (Lonberg (2008) Curr. Opinion20(4):450-459).

The monoclonal antibodies herein specifically include chimericantibodies, humanized antibodies and human antibodies.

Examples of cell binding agents include those agents described for usein WO 2007/085930, which is incorporated herein.

Tumour-associate antigens and cognate antibodies for use in embodimentsof the present invention are listed below, and are described in moredetail on pages 14 to 86 of WO 2017/186894, which is incorporatedherein.

(1) BMPR1B (bone morphogenetic protein receptor-type IB)

(2) E16 (LAT1, SLC7A5)

(3) STEAP1 (six transmembrane epithelial antigen of prostate)

(4) 0772P (CA125, MUC16)

(5) MPF (MPF, MSLN, SMR, megakaryocyte potentiating factor, mesothelin)

(6) Napi3b (NAPI-3B, NPTIIb, SLC34A2, solute carrier family 34 (sodiumphosphate), member 2, type II sodium-dependent phosphate transporter 3b)

(7) Sema 5b (FLJ10372, KIAA1445, Mm.42015, SEMA5B, SEMAG, Semaphorin 5bHlog, sema domain, seven thrombospondin repeats (type 1 and type1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5B)

(8) PSCA hlg (2700050C12Rik, C530008O16Rik, RIKEN cDNA 2700050C12, RIKENcDNA 2700050C12 gene)

(9) ETBR (Endothelin type B receptor)

(10) MSG783 (RNF124, hypothetical protein FLJ20315)

(11) STEAP2 (HGNC_8639, IPCA-1, PCANAP1, STAMP1, STEAP2, STMP, prostatecancer associated gene 1, prostate cancer associated protein 1, sixtransmembrane epithelial antigen of prostate 2, six transmembraneprostate protein)

(12) TrpM4 (BR22450, FLJ20041, TRPM4, TRPM4B, transient receptorpotential cation 5 channel, subfamily M, member 4)

(13) CRIPTO (CR, CR1, CRGF, CRIPTO, TDGF1, teratocarcinoma-derivedgrowth factor)

(14) CD21 (CR2 (Complement receptor 2) or C3DR (C3d/Epstein Barr virusreceptor) or Hs.73792)

(15) CD79b (CD79B, CD79(3, IGb (immunoglobulin-associated beta), B29)

(16) FcRH2 (IFGP4, IRTA4, SPAP1A (SH2 domain containing phosphataseanchor protein 1a), SPAP1B, SPAP1C)

(17) HER2 (ErbB2)

(18) NCA (CEACAM6)

(19) MDP (DPEP1)

(20) IL20R-alpha (IL20Ra, ZCYTOR7)

(21) Brevican (BCAN, BEHAB)

(22) EphB2R (DRT, ERK, Hek5, EPHT3, Tyro5)

(23) ASLG659 (B7h)

(24) PSCA (Prostate stem cell antigen precursor)

(25) GEDA

(26) BAFF-R (B cell-activating factor receptor, BLyS receptor 3, BR3)

(27) CD22 (B-cell receptor CD22-B isoform, BL-CAM, Lyb-8, Lyb8,SIGLEC-2, FLJ22814)

(27a) CD22 (CD22 molecule)

(28) CD79a (CD79A, CD79alpha), immunoglobulin-associated alpha, a Bcell-specific protein that covalently interacts with Ig beta (CD79B) andforms a complex on the surface with Ig M molecules, transduces a signalinvolved in B-cell differentiation), pl: 4.84, MW: 25028 TM: 2 [P] GeneChromosome: 19q13.2).

(29) CXCR5 (Burkitt's lymphoma receptor 1, a G protein-coupled receptorthat is activated by the CXCL13 chemokine, functions in lymphocytemigration and humoral defense, plays a role in HIV-2 infection andperhaps development of AIDS, lymphoma, myeloma, and leukemia); 372 aa,pl: 8.54 MW: 41959 TM: 7 [P] Gene Chromosome: 11q23.3,

(30) HLA-DOB (Beta subunit of MHC class II molecule (Ia antigen) thatbinds peptides and presents them to CD4+T lymphocytes); 273 aa, pl:6.56, MW: 30820.TM: 1 [P] Gene Chromosome: 6p21.3)

(31) P2X5 (Purinergic receptor P2X ligand-gated ion channel 5, an ionchannel gated by extracellular ATP, may be involved in synaptictransmission and neurogenesis, deficiency may contribute to thepathophysiology of idiopathic detrusor instability); 422 aa), pl: 7.63,MW: 47206 TM: 1 [P] Gene Chromosome: 17p13.3).

(32) CD72 (B-cell differentiation antigen CD72, Lyb-2); 359 aa, pl:8.66, MW: 40225, TM: 1 5 [P] Gene Chromosome: 9p13.3).

(33) LY64 (Lymphocyte antigen 64 (RP105), type I membrane protein of theleucine rich repeat (LRR) family, regulates B-cell activation andapoptosis, loss of function is associated with increased diseaseactivity in patients with systemic lupus erythematosis); 661 aa, pl:6.20, MW: 74147 TM: 1 [P] Gene Chromosome: 5q12).

(34) FcRH1 (Fc receptor-like protein 1, a putative receptor for theimmunoglobulin Fc domain that contains C2 type Ig-like and ITAM domains,may have a role in B-lymphocyte differentiation); 429 aa, pl: 5.28, MW:46925 TM: 1 [P] Gene Chromosome: 1q21-1q22)

(35) IRTA2 (Immunoglobulin superfamily receptor translocation associated2, a putative immunoreceptor with possible roles in B cell developmentand lymphomagenesis; deregulation of the gene by translocation occurs insome B cell malignancies); 977 aa, pl: 6.88, MW: 106468, TM: 1 [P] GeneChromosome: 1q21)

(36) TENB2 (TMEFF2, tomoregulin, TPEF, HPP1, TR, putative transmembraneproteoglycan, related to the EGF/heregulin family of growth factors andfollistatin); 374 aa)

(37) PSMA-FOLH1 (Folate hydrolase (prostate-specific membrane antigen)1)

(38) SST (Somatostatin Receptor; note that there are 5 subtypes)

(38.1) SSTR2 (Somatostatin receptor 2)

(38.2) SSTR5 (Somatostatin receptor 5)

(38.3) SSTR1

(38.4) SSTR3

(38.5) SSTR4

AvB6—Both subunits (39+40)

(39) ITGAV (Integrin, alpha V)

(40) ITGB6 (Integrin, beta 6)

(41) CEACAM5 (Carcinoembryonic antigen-related cell adhesion molecule 5)

(42) MET (met proto-oncogene; hepatocyte growth factor receptor)

(43) MUC1 (Mucin 1, cell surface associated)

(44) CA9 (Carbonic anhydrase IX)

(45) EGFRvIII (Epidermal growth factor receptor (EGFR), transcriptvariant 3,

(46) CD33 (CD33 molecule)

(47) CD19 (CD19 molecule)

(48) IL2RA (Interleukin 2 receptor, alpha); NCBI Reference Sequence:NM_000417.2);

(49) AXL (AXL receptor tyrosine kinase)

(50) CD30—TNFRSF8 (Tumor necrosis factor receptor superfamily, member 8)

(51) BCMA (B-cell maturation antigen)—TNFRSF17 (Tumor necrosis factorreceptor superfamily, member 17)

(52) CT Ags—CTA (Cancer Testis Antigens)

(53) CD174 (Lewis Y)—FUT3 (fucosyltransferase 3 (galactoside3(4)-L-fucosyltransferase, Lewis blood group)

(54) CLEC14A (C-type lectin domain family 14, member A; Genbankaccession no. NM175060)

(55) GRP78—HSPA5 (heat shock 70 kDa protein 5 (glucose-regulatedprotein, 78 kDa)

(56) CD70 (CD70 molecule) L08096

(57) Stem Cell specific antigens. For example:

-   -   5T4 (see entry (63) below)    -   CD25 (see entry (48) above)    -   CD32    -   LGR5/GPR49    -   Prominin/CD133

(58) ASG-5

(59) ENPP3 (Ectonucleotide pyrophosphatase/phosphodiesterase 3)

(60) PRR4 (Proline rich 4 (lacrimal))

(61) GCC—GUCY2C (guanylate cyclase 2C (heat stable enterotoxin receptor)

(62) Liv-1—SLC39A6 (Solute carrier family 39 (zinc transporter), member6)

(63) 5T4, Trophoblast glycoprotein, TPBG—TPBG (trophoblast glycoprotein)

(64) CD56—NCMA1 (Neural cell adhesion molecule 1)

(65) CanAg (Tumor associated antigen CA242)

(66) FOLR1 (Folate Receptor 1)

(67) GPNMB (Glycoprotein (transmembrane) nmb)

(68) TIM-1—HAVCR1 (Hepatitis A virus cellular receptor 1)

(69) RG-1/Prostate tumor target Mindin—Mindin/RG-1

(70) B7-H4—VTCN1 (V-set domain containing T cell activation inhibitor 1

(71) PTK7 (PTK7 protein tyrosine kinase 7)

(72) CD37 (CD37 molecule)

(73) CD138—SDC1 (syndecan 1)

(74) CD74 (CD74 molecule, major histocompatibility complex, class IIinvariant chain)

(75) Claudins—CLs (Claudins)

(76) EGFR (Epidermal growth factor receptor)

(77) Her3 (ErbB3)—ERBB3 (v-erb-b2 erythroblastic leukemia viral oncogenehomolog 3

(avian))

(78) RON—MST1R (macrophage stimulating 1 receptor (c-met-relatedtyrosine kinase))

(79) EPHA2 (EPH receptor A2)

(80) CD20—MS4A1 (membrane-spanning 4-domains, subfamily A, member 1)

(81) Tenascin C—TNC (Tenascin C)

(82) FAP (Fibroblast activation protein, alpha)

(83) DKK-1 (Dickkopf 1 homolog (Xenopus laevis)

(84) CD52 (CD52 molecule)

(85) CS1—SLAMF7 (SLAM family member 7)

(86) Endoglin—ENG (Endoglin)

(87) Annexin A1—ANXA1 (Annexin A1)

(88) V-CAM (CD106)—VCAM1 (Vascular cell adhesion molecule 1)

An additional tumour-associate antigen and cognate antibodies ofinterest are:

(89) ASCT2 (ASC transporter 2, also known as SLC1A5).

ASCT2 antibodies are described in WO 2018/089393, which is incorporatedherein by reference

The cell binding agent may be labelled, for example to aid detection orpurification of the agent either prior to incorporation as a conjugate,or as part of the conjugate. The label may be a biotin label. In anotherembodiment, the cell binding agent may be labelled with a radioisotope.

Methods of Treatment

The conjugates of the present invention may be used in a method oftherapy. Also provided is a method of treatment, comprisingadministering to a subject in need of treatment atherapeutically-effective amount of a conjugate of formula IV. The term“therapeutically effective amount” is an amount sufficient to showbenefit to a patient. Such benefit may be at least amelioration of atleast one symptom. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage, is within the responsibility of general practitioners and othermedical doctors.

A conjugate may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated. Examples of treatments and therapies include,but are not limited to, chemotherapy (the administration of activeagents, including, e.g. drugs); surgery; and radiation therapy.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto the active ingredient, i.e. a conjugate of formula IV, apharmaceutically acceptable excipient, carrier, buffer, stabiliser orother materials well known to those skilled in the art. Such materialsshould be non-toxic and should not interfere with the efficacy of theactive ingredient. The precise nature of the carrier or other materialwill depend on the route of administration, which may be oral, or byinjection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carrier oran adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. A capsule may comprise asolid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

The Conjugates can be used to treat proliferative disease and autoimmunedisease. The term “proliferative disease” pertains to an unwanted oruncontrolled cellular proliferation of excessive or abnormal cells whichis undesired, such as, neoplastic or hyperplastic growth, whether invitro or in vivo.

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g., histocytoma, glioma,astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer,gastrointestinal cancer, bowel cancer, colon cancer, breast carcinoma,ovarian carcinoma, prostate cancer, testicular cancer, liver cancer,kidney cancer, bladder cancer, pancreatic cancer, brain cancer, sarcoma,osteosarcoma, Kaposi's sarcoma, melanoma), leukemias, psoriasis, bonediseases, fibroproliferative disorders (e.g. of connective tissues), andatherosclerosis. Other cancers of interest include, but are not limitedto, haematological; malignancies such as leukemias and lymphomas, suchas non-Hodgkin lymphoma, and subtypes such as DLBCL, marginal zone,mantle zone, and follicular, Hodgkin lymphoma, AML, and other cancers ofB or T cell origin. Any type of cell may be treated, including but notlimited to, lung, gastrointestinal (including, e.g. bowel, colon),breast (mammary), ovarian, prostate, liver (hepatic), kidney (renal),bladder, pancreas, brain, and skin.

Examples of autoimmune disease include the following: rheumatoidarthritis, autoimmune demyelinative diseases (e.g., multiple sclerosis,allergic encephalomyelitis), psoriatic arthritis, endocrineophthalmopathy, uveoretinitis, systemic lupus erythematosus, myastheniagravis, Graves' disease, glomerulonephritis, autoimmune hepatologicaldisorder, inflammatory bowel disease (e.g., Crohn's disease),anaphylaxis, allergic reaction, Sjögren's syndrome, type I diabetesmellitus, primary biliary cirrhosis, Wegener's granulomatosis,fibromyalgia, polymyositis, dermatomyositis, multiple endocrine failure,Schmidt's syndrome, autoimmune uveitis, Addison's disease, adrenalitis,thyroiditis, Hashimoto's thyroiditis, autoimmune thyroid disease,pernicious anemia, gastric atrophy, chronic hepatitis, lupoid hepatitis,atherosclerosis, subacute cutaneous lupus erythematosus,hypoparathyroidism, Dressler's syndrome, autoimmune thrombocytopenia,idiopathic thrombocytopenic purpura, hemolytic anemia, pemphigusvulgaris, pemphigus, dermatitis herpetiformis, alopecia arcata,pemphigoid, scleroderma, progressive systemic sclerosis, CREST syndrome(calcinosis, Raynaud's phenomenon, esophageal dysmotility,sclerodactyly, and telangiectasia), male and female autoimmuneinfertility, ankylosing spondolytis, ulcerative colitis, mixedconnective tissue disease, polyarteritis nedosa, systemic necrotizingvasculitis, atopic dermatitis, atopic rhinitis, Goodpasture's syndrome,Chagas' disease, sarcoidosis, rheumatic fever, asthma, recurrentabortion, anti-phospholipid syndrome, farmer's lung, erythemamultiforme, post cardiotomy syndrome, Cushing's syndrome, autoimmunechronic active hepatitis, bird-fancier's lung, toxic epidermalnecrolysis, Alport's syndrome, alveolitis, allergic alveolitis,fibrosing alveolitis, interstitial lung disease, erythema nodosum,pyoderma gangrenosum, transfusion reaction, Takayasu's arteritis,polymyalgia rheumatica, temporal arteritis, schistosomiasis, giant cellarteritis, ascariasis, aspergillosis, Sampter's syndrome, eczema,lymphomatoid granulomatosis, Behcet's disease, Caplan's syndrome,Kawasaki's disease, dengue, encephalomyelitis, endocarditis,endomyocardial fibrosis, endophthalmitis, erythema elevatum et diutinum,psoriasis, erythroblastosis fetalis, eosinophilic faciitis, Shulman'ssyndrome, Felty's syndrome, filariasis, cyclitis, chronic cyclitis,heterochronic cyclitis, Fuch's cyclitis, IgA nephropathy,Henoch-Schonlein purpura, graft versus host disease, transplantationrejection, cardiomyopathy, Eaton-Lambert syndrome, relapsingpolychondritis, cryoglobulinemia, Waldenstrom's macroglobulemia, Evan'ssyndrome, and autoimmune gonadal failure.

In some embodiments, the autoimmune disease is a disorder of Blymphocytes (e.g., systemic lupus erythematosus, Goodpasture's syndrome,rheumatoid arthritis, and type I diabetes), Th1-lymphocytes (e.g.,rheumatoid arthritis, multiple sclerosis, psoriasis, Sjögren's syndrome,Hashimoto's thyroiditis, Graves' disease, primary biliary cirrhosis,Wegener's granulomatosis, tuberculosis, or graft versus host disease),or Th2-lymphocytes (e.g., atopic dermatitis, systemic lupuserythematosus, atopic asthma, rhinoconjunctivitis, allergic rhinitis,Omenn's syndrome, systemic sclerosis, or chronic graft versus hostdisease). Generally, disorders involving dendritic cells involvedisorders of Th1-lymphocytes or Th2-lymphocytes. In some embodiments,the autoimmunie disorder is a T cell-mediated immunological disorder.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.

Examples of chemotherapeutic agents include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine, dichloroplatinum (II), CAS No. 15663-27-1), carboplatin(CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo [4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®,Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, II),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, calicheamicin gamma1l, calicheamicin omegal1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin,marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),pertuzumab (OMNITARG™, 2C4, Genentech), trastuzumab (HERCEPTIN®,Genentech), tositumomab (Bexxar, Corixia), and the antibody drugconjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth). Humanizedmonoclonal antibodies with therapeutic potential as chemotherapeuticagents in combination with the conjugates of the invention include:alemtuzumab, apolizumab, aselizumab, atlizumab, bapineuzumab,bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab,certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab,efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gemtuzumabozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab,matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab,nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab,palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pertuzumab,pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab,resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab,sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tefibazumab,tocilizumab, toralizumab, trastuzumab, tucotuzumab celmoleukin,tucusituzumab, umavizumab, urtoxazumab, and visilizumab.

Formulations

While it is possible for the conjugate to be used (e.g., administered)alone, it is often preferable to present it as a composition orformulation.

In one embodiment, the composition is a pharmaceutical composition(e.g., formulation, preparation, medicament) comprising a conjugate, asdescribed herein, and a pharmaceutically acceptable carrier, diluent, orexcipient.

In one embodiment, the composition is a pharmaceutical compositioncomprising at least one conjugate, as described herein, together withone or more other pharmaceutically acceptable ingredients well known tothose skilled in the art, including, but not limited to,pharmaceutically acceptable carriers, diluents, excipients, adjuvants,fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers,solubilisers, surfactants (e.g., wetting agents), masking agents,colouring agents, flavouring agents, and sweetening agents.

In one embodiment, the composition further comprises other activeagents, for example, other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts. See, for example, Handbook of PharmaceuticalAdditives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (SynapseInformation Resources, Inc., Endicott, N.Y., USA), Remington'sPharmaceutical Sciences, 20th edition, pub. Lippincott, Williams &Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition,1994.

Another aspect of the present invention pertains to methods of making apharmaceutical composition comprising admixing at least one[¹¹C]-radiolabelled conjugate or conjugate-like compound, as definedherein, together with one or more other pharmaceutically acceptableingredients well known to those skilled in the art, e.g., carriers,diluents, excipients, etc. If formulated as discrete units (e.g.,tablets, etc.), each unit contains a predetermined amount (dosage) ofthe active compound.

The term “pharmaceutically acceptable,” as used herein, pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association theactive compound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with carriers(e.g., liquid carriers, finely divided solid carrier, etc.), and thenshaping the product, if necessary.

The formulation may be prepared to provide for rapid or slow release;immediate, delayed, timed, or sustained release; or a combinationthereof.

Formulations suitable for parenteral administration (e.g., byinjection), include aqueous or non-aqueous, isotonic, pyrogen-free,sterile liquids (e.g., solutions, suspensions), in which the activeingredient is dissolved, suspended, or otherwise provided (e.g., in aliposome or other microparticulate). Such liquids may additionallycontain other pharmaceutically acceptable ingredients, such asanti-oxidants, buffers, preservatives, stabilisers, bacteriostats,suspending agents, thickening agents, and solutes which render theformulation isotonic with the blood (or other relevant bodily fluid) ofthe intended recipient. Examples of excipients include, for example,water, alcohols, polyols, glycerol, vegetable oils, and the like.Examples of suitable isotonic carriers for use in such formulationsinclude Sodium Chloride Injection, Ringer's Solution, or LactatedRinger's Injection. Typically, the concentration of the activeingredient in the liquid is from about 1 ng/ml to about 10 μg/ml, forexample from about 10 ng/ml to about 1 μg/ml. The formulations may bepresented in unit-dose or multi-dose sealed containers, for example,ampoules and vials, and may be stored in a freeze-dried (lyophilised)condition requiring only the addition of the sterile liquid carrier, forexample water for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions may be prepared from sterilepowders, granules, and tablets.

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the Conjugates, and compositions comprising the Conjugates,can vary from patient to patient. Determining the optimal dosage willgenerally involve the balancing of the level of therapeutic benefitagainst any risk or deleterious side effects. The selected dosage levelwill depend on a variety of factors including, but not limited to, theactivity of the particular compound, the route of administration, thetime of administration, the rate of excretion of the compound, theduration of the treatment, other drugs, compounds, and/or materials usedin combination, the severity of the condition, and the species, sex,age, weight, condition, general health, and prior medical history of thepatient. The amount of compound and route of administration willultimately be at the discretion of the physician, veterinarian, orclinician, although generally the dosage will be selected to achievelocal concentrations at the site of action which achieve the desiredeffect without causing substantial harmful or deleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the active compound is in the range ofabout 100 ng to about 25 mg (more typically about 1 μg to about 10 mg)per kilogram body weight of the subject per day. Where the activecompound is a salt, an ester, an amide, a prodrug, or the like, theamount administered is calculated on the basis of the parent compoundand so the actual weight to be used is increased proportionately.

The dosage amounts described above may apply to the conjugate or to theeffective amount of compound that is releasable after cleavage of thelinker.

For the prevention or treatment of disease, the appropriate dosage of anADC of the invention will depend on the type of disease to be treated,as defined above, the severity and course of the disease, whether themolecule is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The molecule issuitably administered to the patient at one time or over a series oftreatments. Depending on the type and severity of the disease, about 1μg/kg to 100 mg/kg or more of molecule is an initial candidate dosagefor administration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. Other dosage regimens may be useful. The progress ofthis therapy is easily monitored by conventional techniques and assays.

Drug Loading

The drug loading (p) is the average number of drugs per Ligand unit,which may be a cell binding agent, e.g. antibody.

The average number of drugs per antibody in preparations of ADC fromconjugation reactions may be characterized by conventional means such asUV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, andelectrophoresis. The quantitative distribution of ADC in terms of p mayalso be determined. By ELISA, the averaged value of p in a particularpreparation of ADC may be determined (Hamblett et al (2004) Clin. CancerRes. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852).However, the distribution of p (drug) values is not discernible by theantibody-antigen binding and detection limitation of ELISA. Also, ELISAassay for detection of antibody-drug conjugates does not determine wherethe drug moieties are attached to the antibody, such as the heavy chainor light chain fragments, or the particular amino acid residues. In someinstances, separation, purification, and characterization of homogeneousADC where p is a certain value from ADC with other drug loadings may beachieved by means such as reverse phase HPLC or electrophoresis. Suchtechniques are also applicable to other types of conjugates.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, an antibody may have onlyone or several cysteine thiol groups, or may have only one or severalsufficiently reactive thiol groups through which a linker may beattached. Higher drug loading may cause aggregation, insolubility,toxicity, or loss of cellular permeability of certain antibody-drugconjugates.

Typically, fewer than the theoretical maximum of drug moieties areconjugated to an antibody during a conjugation reaction. An antibody maycontain, for example, many lysine residues that do not react with theDrug Linker. Only the most reactive lysine groups may react with anamine-reactive linker reagent. Also, only the most reactive cysteinethiol groups may react with a thiol-reactive linker reagent. Generally,antibodies do not contain many, if any, free and reactive cysteine thiolgroups which may be linked to a drug moiety. Most cysteine thiolresidues in the antibodies of the compounds exist as disulfide bridgesand must be reduced with a reducing agent such as dithiothreitol (DTT)or TCEP, under partial or total reducing conditions. The loading(drug/antibody ratio) of an ADC may be controlled in several differentmanners, including: (i) limiting the molar excess of Drug Linkerrelative to antibody, (ii) limiting the conjugation reaction time ortemperature, and (iii) partial or limiting reductive conditions forcysteine thiol modification.

Certain antibodies have reducible interchain disulfides, i.e. cysteinebridges. Antibodies may be made reactive for conjugation with linkerreagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol. Reactive thiol groups may be introduced into the antibody(or fragment thereof) by engineering one, two, three, four, or morecysteine residues (e.g., preparing mutant antibodies comprising one ormore non-native cysteine amino acid residues). U.S. Pat. No. 7,521,541teaches engineering antibodies by introduction of reactive cysteineamino acids.

Cysteine amino acids may be engineered at reactive sites in an antibodyand which do not form intrachain or intermolecular disulfide linkages(Junutula, et al., 2008b Nature Biotech., 26(8):925-932; Dornan et al(2009) Blood 114(13):2721-2729; U.S. Pat. Nos. 7,521,541; 7,723,485;WO2009/052249). The engineered cysteine thiols may react with DrugLinkers of the present invention (i.e. of formula I) which havethiol-reactive, electrophilic groups such as maleimide or alpha-haloamides to form ADC with cysteine engineered antibodies. The location ofthe drug unit can thus be designed, controlled, and known. The drugloading can be controlled since the engineered cysteine thiol groupstypically react with drug-linker reagents in high yield. Engineering anIgG antibody to introduce a cysteine amino acid by substitution at asingle site on the heavy or light chain gives two new cysteines on thesymmetrical antibody. A drug loading near 2 can be achieved with nearhomogeneity of the conjugation product ADC.

Where more than one nucleophilic or electrophilic group of the antibodyreacts with Drug Linkers, then the resulting product may be a mixture ofADC compounds with a distribution of drug units attached to an antibody,e.g. 1, 2, 3, etc. Liquid chromatography methods such as polymericreverse phase (PLRP) and hydrophobic interaction (HIC) may separatecompounds in the mixture by drug loading value. Preparations of ADC witha single drug loading value (p) may be isolated, however, these singleloading value ADCs may still be heterogeneous mixtures because the drugunits may be attached, via the linker, at different sites on theantibody.

Thus the antibody-drug conjugate compositions of the invention mayinclude mixtures of antibody-drug conjugates where the antibody has oneor more drug moieties and where the drug moieties may be attached to theantibody at various amino acid residues.

In one embodiment, the average number of drugs per cell binding agent isin the range 1 to 20. In some embodiments the range is selected from 1to 10, 2 to 10, 2 to 8, 2 to 6, and 4 to 10.

In some embodiments, there is one drug per cell binding agent.

General Synthetic Routes

Compounds of formula I where R^(L) is of formula Ia may be synthesisedfrom a compound of Formula 2:

where R^(L*) is -QH by linking a compound of Formula 3:

or an activated version thereof.

Such a reaction may be carried out under amide coupling conditions.

Compounds of Formula 2 may be synthesised by the deprotection of acompound of Formula 4:

where R^(L*prot) is -Q-Prot^(N), where Prot^(N) is an amine protectinggroup.

Compounds of Formula 4 may be synthesised by the coupling of a compoundof Formula 5:

with the compound A3 using the Friedlander reaction.

Compounds of Formula 5 may be synthesised from compounds of Formula 6:

by removal of the trifluoroacetamide protecting group.

Compounds of Formula 6 may be synthesised by coupling: R^(L*prot)—OH tothe compound 17.

Compounds of formula I where R^(L) is of formula Ia or Ib may besynthesised from the compound I11 by coupling of the compound R^(L)—OH,or an activated form thereof.

Amine Protecting Groups

Amine protecting groups are well-known to those skilled in the art.Particular reference is made to the disclosure of suitable protectinggroups in Greene's Protecting Groups in Organic Synthesis, FourthEdition, John Wiley & Sons, 2007 (ISBN 978-0-471-69754-1), pages696-871.

Further Preferences

The following preferences may apply to all aspects of the invention asdescribed above, or may relate to a single aspect. The preferences maybe combined together in any combination.

Q^(X)

In one embodiment, Q is an amino acid residue. The amino acid may be anatural amino acid or a non-natural amino acid.

In one embodiment, Q is selected from: Phe, Lys, Val, Ala, Cit, Leu,Ile, Arg, and Trp, where Cit is citrulline.

In one embodiment, Q comprises a dipeptide residue. The amino acids inthe dipeptide may be any combination of natural amino acids andnon-natural amino acids. In some embodiments, the dipeptide comprisesnatural amino acids. Where the linker is a cathepsin labile linker, thedipeptide is the site of action for cathepsin-mediated cleavage. Thedipeptide then is a recognition site for cathepsin.

In one embodiment, Q is selected from:

-   -   ^(NH)-Phe-Lys-^(C═O),    -   ^(NH)-Val-Ala-^(C═O),    -   ^(NH)-Val-Lys-^(C═O),    -   ^(NH)-Ala-Lys-^(C═O),    -   ^(NH)-Val-Cit-^(C═O),    -   ^(NH)-Phe-Cit-^(C═O),    -   ^(NH)-Leu-Cit-^(C═O),    -   ^(NH)-Ile-Cit-^(C═O),    -   ^(NH)-Phe-Arg-^(C═O),    -   ^(NH)-Trp-Cit-^(C═O), and    -   ^(NH)-Gly-Val-^(C═O);

where Cit is citrulline.

Preferably, Q is selected from:

-   -   ^(NH)-Phe-Lys-^(C═O),    -   ^(NH)-Val-Ala-^(C═O),    -   ^(NH)-Val-Lys-^(C═O),    -   ^(NH)-Ala-Lys-^(C═O), and    -   ^(NH)-Val-Cit-^(C═O).

Most preferably, Q is selected from ^(NH)-Phe-Lys-^(C═O),^(NH)-Val-Cit-^(C═O), or ^(NH)-Val-Ala-^(C═O).

Other dipeptide combinations of interest include:

-   -   ^(NH)-Gly-Gly-^(C═O),    -   ^(NH)-Gly-Val-^(C═O),    -   ^(NH)-Pro-Pro-^(C═O), and    -   ^(NH)-Val-Glu-^(C═O).

Other dipeptide combinations may be used, including those described byDubowchik et al., Bioconjugate Chemistry, 2002, 13,855-869, which isincorporated herein by reference.

In some embodiments, Q is a tripeptide residue. The amino acids in thetripeptide may be any combination of natural amino acids and non-naturalamino acids. In some embodiments, the tripeptide comprises natural aminoacids. Where the linker is a cathepsin labile linker, the tripeptide isthe site of action for cathepsin-mediated cleavage. The tripeptide thenis a recognition site for cathepsin. Tripeptide linkers of particularinterest are:

-   -   ^(NH)-Glu-Val-Ala-^(C═O)    -   ^(NH)-Glu-Val-Cit-^(C═O)    -   ^(NH)-αGlu-Val-Ala-^(C═O)    -   ^(NH)-αGlu-Val-Cit-^(C═O)

In some embodiments, Q is a tetrapeptide residue. The amino acids in thetetrapeptide may be any combination of natural amino acids andnon-natural amino acids. In some embodiments, the tetrapeptide comprisesnatural amino acids. Where the linker is a cathepsin labile linker, thetetrapeptide is the site of action for cathepsin-mediated cleavage. Thetetrapeptide then is a recognition site for cathepsin. Tetrapeptidelinkers of particular interest are:

-   -   ^(NH)-Gly-Gly-Phe-Gly^(C═O); and    -   ^(NH)-Gly-Phe-Gly-Gly^(C═O).

In some embodiments, the tetrapeptide is:

-   -   NH-Gly-Gly-Phe-Gly^(C═O).

In the above representations of peptide residues, ^(NH)- represents theN-terminus, and -^(C═O) represents the C-terminus of the residue. TheC-terminus binds to the NH of A*.

Glu represents the residue of glutamic acid, i.e.:

αGlu represents the residue of glutamic acid when bound via the α-chain,i.e.:

In one embodiment, the amino acid side chain is chemically protected,where appropriate. The side chain protecting group may be a group asdiscussed above. Protected amino acid sequences are cleavable byenzymes. For example, a dipeptide sequence comprising a Boc sidechain-protected Lys residue is cleavable by cathepsin.

Protecting groups for the side chains of amino acids are well known inthe art and are described in the Novabiochem Catalog, and as describedabove.

G^(L)

G^(L) may be selected from

where Ar represents a C₅₋₆ arylene group, e.g. phenylene, and Xrepresents C₁₋₄ alkyl.

In some embodiments, G^(L) is selected from G^(L1-1) and G^(L1-2). Insome of these embodiments, G^(L) is G^(L1-1).

G^(LL)

G^(LL) may be selected from:

where Ar represents a C₅₋₆ arylene group, e.g. phenylene and Xrepresents C₁₋₄ alkyl.

In some embodiments, G^(LL) is selected from G^(LL1-1) and G^(LL1-2). Insome of these embodiments, G^(LL) is G^(LL1-1).

X

X is:

where a=0 to 5, b1=0 to 16, b2=0 to 16, c=0 or 1, d=0 to 5, wherein atleast b1 or b2=0 and at least c1 or c2=0.

a may be 0, 1, 2, 3, 4 or 5. In some embodiments, a is 0 to 3. In someof these embodiments, a is 0 or 1. In further embodiments, a is 0.

b1 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. Insome embodiments, b1 is 0 to 12. In some of these embodiments, b1 is 0to 8, and may be 0, 2, 3, 4, 5 or 8.

b2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. Insome embodiments, b2 is 0 to 12. In some of these embodiments, b2 is 0to 8, and may be 0, 2, 3, 4, 5 or 8.

Only one of b1 and b2 may not be 0.

c1 may be 0 or 1.

c2 may be 0 or 1.

Only one of c1 and c2 may not be 0.

d may be 0, 1, 2, 3, 4 or 5. In some embodiments, d is 0 to 3. In someof these embodiments, d is 1 or 2. In further embodiments, d is 2. Infurther embodiments, d is 5.

In some embodiments of X, a is 0, b1 is 0, c1 is 1, c2 is 0 and d is 2,and b2 may be from 0 to 8. In some of these embodiments, b2 is 0, 2, 3,4, 5 or 8.

In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 0 and d is 0,and b1 may be from 0 to 8. In some of these embodiments, b1 is 0, 2, 3,4, 5 or 8.

In some embodiments of X, a is 0, b1 is 0, c1 is 0, c2 is 0 and d is 1,and b2 may be from 0 to 8. In some of these embodiments, b2 is 0, 2, 3,4, 5 or 8.

In some embodiments of X, b1 is 0, b2 is 0, c1 is 0, c2 is 0 and one ofa and d is 0. The other of a and d is from 1 to 5. In some of theseembodiments, the other of a and d is 1. In other of these embodiments,the other of a and d is 5.

In some embodiments of X, a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, andb1 may be from 0 to 8.

In some of these embodiments, b2 is 0, 2, 3, 4, 5 or 8.

In some embodiments, R^(L) is of formula Ib.

In some embodiments, R^(LL) is formula Ib′.

R^(L1) and R^(L2) are independently selected from H and methyl, ortogether with the carbon atom to which they are bound form acyclopropylene or cyclobutylene group.

In some embodiments, both R^(L1) and R^(L2) are H.

In some embodiments, R^(L1) is H and R^(L2) is methyl.

In some embodiments, both R^(L1) and R^(L2) are methyl.

In some embodiments, R^(L1) and R^(L2) together with the carbon atom towhich they are bound form a cyclopropylene group.

In some embodiments, R^(L1) and R^(L2) together with the carbon atom towhich they are bound form a cyclobutylene group.

In the group Ib, in some embodiments, e is 0. In other embodiments, e is1 and the nitro group may be in any available position of the ring. Insome of these embodiments, it is in the ortho position. In others ofthese embodiments, it is in the para position.

In some embodiments of the fifth aspect of the invention, theenantiomerically enriched form has an enantiomeric ratio greater than60:40, 70:30; 80:20 or 90:10. In further embodiments, the enantiomericratio is greater than 95:5, 97:3 or 99:1.

In some embodiments, R^(L) is selected from:

In some embodiments, R^(LL) is a group derived from the R^(L) groupsabove.

In one embodiment of the first aspect of the invention, the compound offormula I is:

Further Preferences

In some embodiments, the compound of formula I is of the formula I^(P):

and salts and solvates thereof, wherein R^(LP) is a linker forconnection to a cell binding agent, which is selected from:

-   -   (ia):

-   -   wherein    -   Q^(P) is:

-   -   where Q^(XP) is such that Q^(P) is an amino-acid residue, a        dipeptide residue or a tripeptide residue;    -   X^(P) is:

-   -   where aP=0 to 5, bP=0 to 16, cP=0 or 1, dP=0 to 5;    -   G^(L) is a linker for connecting to a Ligand Unit;    -   (ib):

-   -   where R^(L1) and R^(L2) are independently selected from H and        methyl, or together with the carbon atom to which they are bound        form a cyclopropylene or cyclobutylene group; and    -   e is 0 or 1.

aP may be 0, 1, 2, 3, 4 or 5. In some embodiments, aP is 0 to 3. In someof these embodiments, aP is 0 or 1. In further embodiments, aP is 0.

bP may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16. Insome embodiments, b is 0 to 12. In some of these embodiments, bP is 0 to8, and may be 0, 2, 4 or 8.

cP may be 0 or 1.

dP may be 0, 1, 2, 3, 4 or 5. In some embodiments, dP is 0 to 3. In someof these embodiments, dP is 1 or 2. In further embodiments, dP is 2.

In some embodiments of X^(P), aP is 0, cP is 1 and dP is 2, and bP maybe from 0 to 8. In some of these embodiments, bP is 0, 4 or 8.

The preferences for Q^(X) above for compounds of Formula I may apply toQ^(XP), where appropriate.

The preferences for G^(L), R^(L1), R^(L2) and e above for compounds ofFormula I may apply to compounds of Formula I^(P).

In some embodiments, the conjugate of formula IV is of the formulaIV^(P):

L-(D^(LP))_(P)  (IV^(P))

or a pharmaceutically acceptable salt or solvate thereof, wherein L is aLigand unit (i.e., a targeting agent), D^(LP) is a Drug Linker unit thatis of formula III^(P):

R^(LLP) is a linker connected to the Ligand unit selected from

(ia′):

where Q^(P) and X^(P) are as defined above and G^(LL) is a linkerconnected to a Ligand Unit; and

(ib′):

where R^(L1) and R^(L2) are as defined above; and

p is an integer of from 1 to 20.

In some embodiments, the compound of formula I is of the formula I^(P2):

and salts and solvates thereof, wherein R^(LP2) is a linker forconnection to a cell binding agent, which is selected from:

-   -   (ia):

-   -   wherein    -   Q is:

where Q^(X) is such that Q is an amino-acid residue, a dipeptideresidue, a tripeptide residue or a tetrapeptide residue;

-   -   X^(P) is:

-   -   where aP2=0 to 5, b1P2=0 to 16, b2P2=0 to 16, cP2=0 or 1, dP2=0        to 5, wherein at least b1P2 or b2P2=0 (i.e. only one of b1 and        b2 may not be 0);    -   G^(L) is a linker for connecting to a Ligand Unit;    -   (ib):

-   -   where R^(L1) and R^(L2) are independently selected from H and        methyl, or together with the carbon atom to which they are bound        form a cyclopropylene or cyclobutylene group; and    -   e is 0 or 1.

aP2 may be 0, 1, 2, 3, 4 or 5. In some embodiments, aP2 is 0 to 3. Insome of these embodiments, aP2 is 0 or 1. In further embodiments, aP2 is0.

b1P2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.In some embodiments, b1P2 is 0 to 12. In some of these embodiments, b1P2is 0 to 8, and may be 0, 2, 3, 4, 5 or 8.

b2P2 may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.In some embodiments, b2P2 is 0 to 12. In some of these embodiments, b2P2is 0 to 8, and may be 0, 2, 3, 4, 5 or 8.

Only one of b1P2 and b2P2 may not be 0.

cP2 may be 0 or 1.

dP2 may be 0, 1, 2, 3, 4 or 5. In some embodiments, dP2 is 0 to 3. Insome of these embodiments, dP2 is 1 or 2. In further embodiments, dP2 is2. In further embodiments, dP2 is 5.

In some embodiments of X^(P2), aP2 is 0, b1P2 is 0, cP2 is 1 and dP2 is2, and b2P2 may be from 0 to 8. In some of these embodiments, b2P2 is 0,2, 3, 4, 5 or 8.

In some embodiments of X^(P2), aP2 is 1, b2P2 is 0, cP2 is 0 and dP2 is0, and b1P2 may be from 0 to 8. In some of these embodiments, b1P2 is 0,2, 3, 4, 5 or 8.

In some embodiments of X^(P2), aP2 is 0, b1P2 is 0, cP2 is 0 and dP2 is1, and b2P2 may be from 0 to 8. In some of these embodiments, b2P2 is 0,2, 3, 4, 5 or 8.

In some embodiments of X^(P2), b1P2 is 0, b2P2 is 0, cP2 is 0 and one ofaP2 and dP2 is 0.

The other of aP2 and d is from 1 to 5. In some of these embodiments, theother of aP2 and d is 1. In other of these embodiments, the other of aP2and dP2 is 5.

The preferences for Q^(X) above for compounds of Formula I may apply toQ^(X) in Formula Ia^(P2), where appropriate.

The preferences for G^(L), R^(L1), R^(L2) and e above for compounds ofFormula I may apply to compounds of Formula I^(P2).

In some embodiments, the conjugate of formula IV is of the formulaIV^(P2):

L-(D^(LP2))_(p)  (IV^(P2))

or a pharmaceutically acceptable salt or solvate thereof, wherein L is aLigand unit (i.e., a targeting agent), D^(LP2) is a Drug Linker unitthat is of formula III^(P2):

R^(LLP2) is a linker connected to the Ligand unit selected from

(ia′):

where Q and X^(P2) are as defined above and G^(LL) is a linker connectedto a Ligand Unit; and

(ib′):

where R^(L1) and R^(L2) are as defined above; and

p is an integer of from 1 to 20.

EXAMPLES

General Information

Flash chromatography was performed using a Biotage® Isolera™ andfractions checked for purity using thin-layer chromatography (TLC). TLCwas performed using Merck Kieselgel 60 F254 silica gel, with fluorescentindicator on aluminium plates. Visualisation of TLC was achieved with UVlight.

Extraction and chromatography solvents were bought and used withoutfurther purification from VWR U.K.

All fine chemicals were purchased from Sigma-Aldrich unless otherwisestated.

Pegylated reagents were obtained from Quanta biodesign US via StratechUK.

LC/MS Conditions

Method A

Positive mode electrospray mass spectrometry was performed using aWaters Aquity H-class SQD2.

Mobile phases used were solvent A (water with 0.1% formic acid) andsolvent B (acetonitrile with 0.1% formic acid). Initial composition 5% Bheld over 25 seconds, then increased from 5% B to 100% B over a 1 minute35 seconds' period. The composition was held for 50 seconds at 100% B,then returned to 5% B in 5 seconds and held there for 5 seconds. Thetotal duration of the gradient run was 3.0 minutes. Flow rate was 0.8mL/minute. Detection was at 254 nm. Columns: Waters Acquity UPLC® BEHShield RP18 1.7 μm 2.1×50 mm at 50° C. fitted with Waters Acquity UPLC®BEH Shield RP18 VanGuard Pre-column, 130A, 1.7 μm, 2.1 mm×5 mm.

Method B

The HPLC (Waters Alliance 2695) was run using a mobile phase of water(A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%).

Initial composition 5% B held over 25 seconds, then increased from 5% Bto 100% B over a 1 minute 35 seconds' period. The composition was heldfor 50 seconds at 100% B, then returned to 5% B in 5 seconds and heldthere for 5 seconds. The total duration of the gradient run was 3.0minutes. Flow rate was 0.8 mL/minute. Wavelength detection range: 190 to800 nm. Columns: Waters Acquity UPLC® BEH Shield RP18 1.7 μm 2.1×50 mmat 50° C. fitted with Waters Acquity UPLC® BEH Shield RP18 VanGuardPre-column, 130A, 1.7 μm, 2.1 mm×5 mm.

Method C

The HPLC (Waters Alliance 2695) was run using a mobile phase of water(A) (formic acid 0.1%) and acetonitrile (B) (formic acid 0.1%).

Initial composition 5% B held over 1 min, then increase from 5% B to100% B over a 9 min period. The composition was held for 2 min at 100%B, then returned to 5% B in 0.10 minutes and hold there for 3 min. Totalgradient run time equals 15 min. Flow rate 0.6 mL/min. Wavelengthdetection range: 190 to 800 nm. Oven temperature: 50° C. Column: ACEExcel 2 C18-AR, 2μ, 3.0×100 mm.

HPLC Conditions

Reverse-phase ultra-fast high-performance liquid chromatography (UFLC)was carried out on a Shimadzu Prominence™ machine using a Phenomenex™Gemini NX 5μ C18 column (at 50° C.) dimensions: 150×21.2 mm. Eluentsused were solvent A (H₂O with 0.1% formic acid) and solvent B (CH₃CNwith 0.1% formic acid). All UFLC experiments were performed withgradient conditions: Initial composition 13% B increased to 30% B over a3 minutes period, then increased to 45% B over 8 minutes and again to100% over 6 minutes before returning to 13% over 2 min and hold for 1min. The total duration of the gradient run was 20.0 minutes. Flow ratewas 20.0 mL/minute and detection was at 254 and 223 nm.

NMR Method

Proton NMR chemical shift values were measured on the delta scale at 400MHz using a Bruker AV400. The following abbreviations have been used: s,singlet; d, doublet; t, triplet; q, quartet; quin, quintet; m,multiplet; br, broad. Coupling constants are reported in Hz.

Synthesis of Key Intermediates

a) N-(5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (I2)

5,6,7,8-tetrahydronaphthalen-1-amine I1 (8.54 g, 58.0 mmol) wasdissolved in dichloromethane (80 mL). Triethylamine (18 mL, 129 mmol)was added and the mixture cooled to 0° C. Dropwise, acetic anhydride(11.5 mL, 122 mmol) was added, upon completion of the addition, thereaction mixture was warmed to rt and stirred for 45 min, whereupon LCMSindicated the reaction was complete. The mixture was diluted withCH₂Cl₂, washed with H₂O, sat. NaHCO₃, 10% citric acid, the organic phasedried over MgSO₄ and concentrated in vacuo. The off-white solid wastriturated with 1:3 Et₂O/isohexane to afford I1 (10.8 g, 57.1 mmol, 98%Yield) as a white solid which was used without further purification.LC/MS (method A): retention time 1.44 mins (ES+) m/z 190 [M+H]⁺

b) N-(4-nitro-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (I3)

N-(5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I2 (1.00 g, 5.2840 mmol)was added portion-wise to sulfuric acid (15 mL, 281 mmol) at −5° C.Sodium nitrate (450 mg, 5.2945 mmol) was added portion-wise to thereaction mixture and stirred for 30 min at −5° C. whereupon LCMSindicated no further reaction progress. The reaction mixture was pouredonto ice with external cooling, the aqueous mixture extracted withCH₂Cl₂, the organic phase dried over MgSO₄ and purified by Isolera(10-80% EtOAc in isohexane) to afford a mixture ofN-(4-nitro-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I3 andN-(2-nitro-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (956 mg, 4.0811mmol, 77% Yield) as a white/yellow solid. LC/MS (method A): retentiontime 1.53 mins (ES+) m/z 235 [M+H]⁺.

c) N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (I4)

N-(4-nitro-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I3 (1.01 g, 4.31mmol) was dissolved in acetone (30 mL). Magnesium sulfate in water (3.9mL, 5.9 mmol, 1.5 mol/L) was added and the mixture was cooled to 0° C.Potassium permanganate (2.07 g, 13.0 mmol) was added portionwise to thereaction mixture and the mixture warmed to rt and stirred for 50 min,whereupon TLC indicated the reaction was complete. The reaction mixturewas filtered through Celite, the solids washed with CHCl₃ and theresulting organic mixture washed with H₂O, brine, dried over MgSO₄ andpurified by isolera (20-50% EtOAc in isohexane) to afford a mixture ofN-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide 14 andN-(2-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (709 mg,2.86 mmol, 66%) as a white/yellow solid. LC/MS (method A): retentiontime 1.44 mins (ES+) m/z 190 [M+H]⁺

d) 8-amino-5-nitro-3,4-dihydronaphthalen-1(2H)-one (15)

A mixture ofN-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide I4 andN-(2-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (709 mg,2.8561 mmol) and 6N hydrochloric acid (7 mL) were stirred at 80° C. for2.5 h, whereupon LCMS indicated the reaction was complete. The reactionmixture was cooled in an ice bath and 6N NaOH solution was added untilthe pH was basic. The aqueous mixture was extracted with CH₂Cl₂, theorganic phase dried over MgSO₄ and concentrated in vacuo. Isolera (0-50%EtOAc in isohexane) afforded8-amino-5-nitro-3,4-dihydronaphthalen-1(2H)-one 15 (320 mg, 1.552 mmol,54% Yield) as a yellow/orange solid. LC/MS (method A): retention time1.54 mins (ES+) m/z 207 [M+H]⁺

e)2,2,2-trifluoro-N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide(I6)

8-amino-5-nitro-3,4-dihydronaphthalen-1(2H)-one I5 (430 mg, 2.0854 mmol)was dissolved in dichloromethane (20 mL). Pyridine (340 μL, 4.20 mmol)was added and the mixture cooled to 0° C. Trifluoroacetic anhydride (590μL, 4.197 mmol) was added and stirred for 30 min, whereupon LCMSindicated the reaction was complete. The mixture was diluted withCH₂Cl₂, washed with H₂O, the organic phase dried over MgSO₄ andconcentrated in vacuo to afford2,2,2-trifluoro-N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamideI6 (630 mg, 2.0846 mmol, >99% Yield) as a yellow solid, which was usedwithout further purification. LC/MS (method A): retention time 1.86 min(ES+) m/z 301X [M−H]⁻

f)N-(4-amino-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)-2,2,2-trifluoroacetamide(I7)

Zinc (2.73 g, 41.7 mmol) was suspended in methanol (80 mL), formic acid(4 mL) and water (4 mL) and the mixture cooled to 0° C.2,2,2-trifluoro-N-(4-nitro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamideI6 (568 mg, 2.0865 mmol) was added portion-wise and the mixture stirredat 0° C. for 30 min, whereupon LCMS indicated the reaction was complete.The reaction mixture was filtered, the filtrate diluted with EtOAc andwashed with sat NaHCO₃. The organic phase was dried over MgSO₄ andconcentrated in vacuo to affordN-(4-amino-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)-2,2,2-trifluoroacetamide17 (568 mg, 2.0865 mmol, >99% Yield) as a yellow solid, which was usedwithout further purification. LC/MS (method A): retention time 1.65 min(ES+) m/z 273 [M+H]⁺

g)N-(4-acetamido-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)-2,2,2-trifluoroacetamide(I8)

N-(8-amino-4-oxo-tetralin-5-yl)-2,2,2-trifluoro-acetamide I7 (568 mg,2.0865 mmol) was dissolved in dichloromethane (20 mL). Triethylamine(580 μL, 4.16 mmol) then acetyl chloride (297 μL, 4.173 mmol) were addedand the mixture stirred for 30 min, whereupon LCMS indicated thereaction was complete. The reaction mixture was diluted with CH₂Cl₂,washed with H₂O, the organic phase dried over MgSO₄ and concentrated invacuo to affordN-(8-acetamido-4-oxo-tetralin-5-yl)-2,2,2-trifluoro-acetamide 18 (655mg, 2.084 mmol, >99% yield) as a yellow solid, which was used withoutfurther purification. LC/MS (method A): retention time 1.55 min (ES+)m/z 315 [M+H]⁺

h) N-(4-amino-5-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (I9)

N-(8-acetamido-4-oxo-tetralin-5-yl)-2,2,2-trifluoro-acetamide I8 (2.77g, 8.81 mmol) was dissolved in methanol (240 mL) and water (17 mL).Potassium carbonate (4.88 g, 35.3 mmol) was added and the mixturestirred for 1.5 h at 50° C., whereupon LCMS indicated the reaction wascomplete. The reaction mixture was cooled, concentrated in vacuo,dissolved in 10% MeOH in CH₂C₁₂ and washed with H₂O. The organic phasewas dried over MgSO₄ and purified by isolera chromatography (2-15% MeOHin CH₂Cl₂) to afford N-(8-amino-1-oxo-tetralin-5-yl)acetamide 19 (1.20g, 5.50 mmol, 62.3% Yield) as a yellow solid. LC/MS (method A):retention time 0.98 min (ES+) m/z 219 [M+H]⁺

i)(S)—N-(9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzoldepyrano[3,4′:6,7]indolizino[1,2-b]quinolin-4-yl)acetamide(I10)

N-(8-amino-1-oxo-tetralin-5-yl)acetamide I9 (641 mg, 2.94 mmol, 1.0eq.),(S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trioneA3 (840 mg, 3.19 mmol, 1.1 eq.) and PPTS (740 mg, 2.95 mmol, 1.0 eq.)were dissolved in toluene (60 mL) and stirred at reflux for 3 h,whereupon LCMS indicated 19 had been consumed. The reaction mixture wascooled and concentrated in vacuo. The resulting solids were trituratedwith acetonitrile, then acetone to afford 110 as a brown solid withminor TsOH contamination (1.26 g, 96%). LC/MS (method A): retention time1.32 mins (ES+) m/z 447 [M+H]⁺

j)(S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzoldepyrano[3,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione(111)

(S)—N-(9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)acetamide(I10) (1.26 g, 2.83 mmol, 1.0 eq.) was dissolved in hydrochloric acid (6mol/L) in H₂O (12 mL) and the mixture stirred for 5 h at 80° C.,whereupon LCMS indicated 110 had been consumed. The reaction mixture wasdiluted with H₂O and concentrated in vacuo to afford(S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione I11(1.51 g, 2.85 mmol, 90 mass %, 101% Yield) as a red crystaline solid.LC/MS (method A): retention time 1.36 mins (ES+) m/z 405 [M+H]⁺.

Alternate Synthesis of I11

IPC, Purity and Assay Method for this Synthesis

a) 5-bromo-8-nitro-tetralin-1-one (I13)

A solution of potassium nitrate (1.15 eq., 13.83 g) dissolved insulphuric acid (Conc., 5.0 rel. vol., 160 mL), was added (addition time4-12 h, maintaining the temperature below 10° C.) to a solution of5-bromotetralin-1-one (I12)(1.0 equiv., 26.77 g) in sulfuric acid(Conc., 5.0 rel. vol., 160 mL) under nitrogen. When the reaction wascomplete the reaction mixture was transferred to flask containing water(36 rel. vol., 1.15 L) adjusting the transfer rate to keep thetemperature below 10° C. The resulting solid was filtered, washed withwater (4.0 rel. vol., 128 mL) three times and then dried at ˜40° C. for24 h. The dry cake was dissolved in a mixture of acetone (2.5 rel. vol.,80 mL) and water (0.38 rel. vol., 12.2 mL) heated to ˜75° C. and thencooled to ˜20° C. The resulting solid was removed by filtration. Thesolvent was swapped to ethanol by distillation and the solution volumereduced to a 2.0 rel. vol. (64 mL). The solution was cooled to ˜25° C.and the resulting solid collected by filtration. The solid was washedwith ethanol (1.0 Rel. Vol., 32 mL) then dried under vacuum at 40° C. togive 5-bromo-8-nitro-tetralin-1-one I13 (15.36 g, 40%) as a brown solid;RT 14.0 min

Method 1 IPC, Purity and Assay Method for Bromo-8-Nitro-Tetralin-1-One.

b) N-(8-nitro-1-oxo-tetralin-5-yl)acetamide (I14)

A solution of bromo-8-nitro-tetralin-1-one (I13)(1.0 eq., 18.0 g, 90.6%ww), acetamide (1.2 eq., 4.72 g),tris(dibenzylideneacetone)dipalladium(0) (0.01 eq., 0.61 g) andpotassium phosphate (1.4 eq., 19.8 g) in dioxane (15 rel. vol., 270 mL)under nitrogen was heated to ˜70° C. When the reaction was complete thesolution was cooled to ˜20° C. and diluted with dioxane (5 rel. vol.,90.0 mL) and filtered. The solvent was swapped to ethanol and the volumereduced to a total reaction volume of 3 rel. vol. (54.0 mL). thesolution was cooled to ˜20° C. and the resulting solid collected byfiltration and washed with MTBE (methyl tert-butyl ether)(1.0 rel. vol.,18.0 mL). The solid was dried under vacuum at 40° C. to giveN-(8-nitro-1-oxo-tetralin-5-yl)acetamide I14 (10.0 g, 60.6%) as a darkyellow solid; RT 8.86 min.

c) N-(8-amino-1-oxo-tetralin-5-yl)acetamide (I15)

Palladium hydroxide on carbon (20% w/w, 0.15 eq., 5.25 g) was added to asolution of N-(8-nitro-1-oxo-tetralin-5-yl)acetamide (I14)(1.0 eq., 32.6g) in methanol (40 rel. vol., 1250 mL). The reaction mixture was placedunder a hydrogen atmosphere at ˜40 psi, at ˜40° C. for 8 h. The hydrogenwas removed and replaced with nitrogen and the catalyst was removed byfiltration over cellulose, washing the cellulose with methanol (4.0 rel.vol., 130 mL). The solution volume was reduced to 4.0 rel. vol. bydistillation and then diluted with MTBE (4 rel. vol, 130 mL). Theresulting solid was collect by filtration, washed with MTBE (2 rel.vol., 65 mL) and dried under vacuum at 40° C. to giveN-(8-amino-1-oxo-tetralin-5-yl)acetamide I15 (21.1 g, 77.8%) as a greygreen solid; RT 5.44 min.

d) 5,8-diaminotetralin-1-one (I16)

A solution of N-(8-amino-1-oxo-tetralin-5-yl)acetamide (I15)(1.0 eq.,10.0 g) in hydrochloric acid (5M, 6.0 rel. vol., 60 mL), was held at˜90° C. for 3 h. The temperature was reduced to 25° C. and sodiumhydroxide (2M, 4.0 rel. vol., 40 mL) was added until pH 10.0 wasachieved, maintaining the temperature 25° C. The resulting solid wascollected by filtration and washed with water (2.0 rel. vol., 20 mL).The wet cake was dissolved in tetrahydrofuran (60 rel. vol., 600 mL) andfiltered. The solution was concentrated to 5.0 rel. vol. and heptane (20rel. vol., 200 mL) added. The solution was concentrated to 10.0 rel.vol. and further heptane (20 rel. vol., 200 mL) added, and then thevolume reduce dto 10.0 rel. vol. again. The resulting solid wascollected by filtration and washed with heptane (5.0 rel. vol., 50 mL).The solid was dried under vacuum at 40° C. for 17 h to give5,8-diaminotetralin-1-one (116)(6.90 g, 82.7%) as a yellow solid; 1H NMR(400 MHz DMSO-d6) δ ppm 1.82 (m, 2H), 2.38 (t, J=2.0 Hz, 2H), 2.47 (t,J=2.0 Hz, 2H), 6.34 (d, J=2.0 Hz, 1H), 6.68 (d, J=2.0 Hz, 1H); RT 3.90

e)(S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione(I11)

A solution of 5,8-diaminotetralin-1-one (I16)(1.0 eq., 5.0 g),(4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10-trione(A3)(1.06 eq., 7.9 g), and pyridinium para-toluenesulfonate (1.0 eq.,7.2 g) in toluene (50.0 rel. vol., 250 mL) was held at 120° C. for 15 h.The volume of the solution was reduced to 2.0 rel. vol. and then dilutedwith acetonitrile (20 rel. vol., 100 mL) and water (20 rel. vol., 100mL). The resulting slurry was filtered and the solid washed with aqueousacetonitrile (1:1, 20 rel. vol., 100 mL). The solid was slurried withaqueous methanol (water:MeOH 3:1, 40 rel. vol., 200 mL), filtered andwashed with aqueous methanol (1:1, 20 rel. vol., 100 mL). The solid wasslurried with water (60 rel. vol., 300 mL) at 50° C., filtered andwashed with water (10 rel. vol., 50 mL). The solid was slurried withaqueous acetonitrile (water:acetonitrile, 1:3, 40 rel. vol., 200 mL) at30° C., filtered and washed with aqueous acetonitrile(water:acetonitrile, 1:3, 5 rel. vol., 50 mL) and then dried undervacuum at 40° C. to give(S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione(I11) as white solid (5.0 g, 43.7%); RT 5.13.

Synthesis of I18

a) tert-butyl(5)-(2-((2-((1-((2-((4-amino-5-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)carbamate(I17)

Boc-GGFG-OH (227 mg, 0.52 mmol) and EEDQ (157 mg, 0.634 mmol) weresolubilised in CH₂Cl₂ (25 mL) and the mixture stirred for 15 min, untilthe peptide has gone into solution. Compound I16 (100 mg, 0.56747 mmol)was subsequently added and the mixture left to stir until complete.After 1 h, the reaction looked 90% complete by LVMC. The mixture hasgone thicker as the product is crashing out. The mixture was left foranother hour before vaccing down to dryness. The crude was taken up inEt₂O (50 mL). The solid was filtered and subsequently taken up in CH₂Cl₂(50 mL) to purify further. The solid was filtered and dried to giveproduct I17 (273 mg, 0.459 mmol, 80.9% Yield) as a grey solid.Analytical data: LCMS 3 min: ES+=1.46 min, m/z 595.7 [M+H]⁺.

b)(S)-2-(2-(2-aminoacetamido)acetamido)-N-(2-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-2-oxoethyl)-3-phenylpropanamide(I18)

Aniline 117 (450 mg, 1.045 mMol), lactone A5 (280 mg, 1.064 mMol) andpyridinium p-toluenesulfonate (273 mg, 1.086 mMol) were solubilised intoluene (20 mL) and the mixture was heated to 150° C. (high reflux).MeOH (4 mL) was added to help solubilise the mixture. After 7 h thecrude reaction was vacced down to dryness. The crude product waspurified by silica gel chromatography (CHCl₃/MeOH, 100% to 65:35) togive product I18 (259 mg, 0.359 mMol, 78.1 yield). Analytical data: LCMS3 min: ES+=1.17 min, m/z 722.8 [M+H]⁺.

Alternative Synthesis of I16

a) 5-Fluoro-8-nitro-tetralin-1-one (I20)

5-fluorotetralin-1-one 119 (4.7 g, 29 mmol) was solubilised in ½ theamount of sulfuric acid (120 mL) in a 3 neck round bottom flask. Themixture was stirred until all the solid has dissolved and then cooled to0-5° C. In a dropping funnel, dissolve potassium nitrate (3 g, 29.6730mmol) into the remaining half of sulfuric acid (120 mL) at 0-5° C.Slowly add to the SM mixture making sure to maintain the solution cool(45 min). Stir at 0-5° C. until complete. The reaction mixture wassubsequently quenched with water (250 mL) and left to stir at 0-5° C.The solid was filtered and washed with water (50 mL). The solid wasdried in a vacuum oven for 2 h at 50° C. The crude solid was slurried inEt₂O overnight before being cooled to 0° C. and filtered. The wet cakewas washed with more cold Et₂O (50 mL) and left to dry in a vacuum ovenat 50° C. to give pure product 120 (5.5 g, 26 mmol, 92% yield) as alight pink fine powder. LCMS (Method B): ES+=1.55 min, m/z 210.1 [M+H]⁺.

b) 5-Amino-8-nitro-tetralin-1-one (I21)

Compound I20 (2.7 g, 13 mmol) was solubilised in CH₃CN (2.5 mL) andNH₄OH (21 mass %) in H₂O (8 mL, 40 mmol) was added to a sealed pressureresistant tube and heated to 185° C. Once complete, the mixture wastransferred to a round bottom flask and vacced down. The crude waspurified by silica gel column chromatography (CHCl₃/MeOH; 100 to 99:1)to give pure product 121 (1.1 g, 5.3 mmol, 41% yield) as a black solid.LCMS (Method B): ES⁺=1.34 min, m/z 207.1 [M+H]⁺.

c) 5,8-diaminotetralin-1-one (I16)

Compound I21 (1.35 g, 6.55 mmol) was dissolved in a mixture of methanol(20 mL), H₂O (1 mL) and formic acid (1 mL) at 0° C. Zinc (8.5 g, 130mmol) was slowly added, making sure to keep the temperature below 40° C.A little more formic acid/H₂O (0.5 mL) was added to push the reaction tocompletion. The reaction mixture was filtered, and the filtrate dilutedwith EtOAc and CH₂Cl₂ before being vacced down. The crude was dryloading onto silica gel column chromatography (CHCl₃/EtOAc; 100 to 7:3then CHCl₃/MeOH; 99:1 to 98:2) to give pure product I16 (1.015 g, 5.760mmol, 88.0% Yield). LCMS (Method B): ES⁺=0.2 min, m/z not observed.

Example 1

a) Allyl((S)-3-methyl-1-oxo-1-(((S)-1-oxo-1-((5-oxo-4-(2,2,2-trifluoroacetamido)-5,6,7,8-tetrahydronaphthalen-1-yl)amino)propan-2-yl)amino)butan-2-yl)carbamate(A1)

DCC (6.54 g, 31.7 mMol) and HOPO (3.36 g, 30.2 mMol) were added to asolution of alloc-Val-Ala-OH (9.09 g, 31.7 mmol) and 17 (7.85 g, 28.8mMol) in CH₂Cl₂ (300 mL) at 25° C. The resulting mixture was left tostir overnight. The white solid that formed during the reaction wasfiltered out and washed with cold CH₂Cl₂. The filtrate was washed withwater (150 mL) and brine (150 mL). The organic layer was dried overMgSO₄, filtered and evaporated. The crude product was purified by silicagel chromatography (Hex/EtOAc, 60:40). Product A1 isolated wascontaminated with co-eluting DCU (21.1 g, 140% yield). LC/MS (Method B):ES⁺=1.81 min, m/z 527.6 [M+H]⁺.

b)Allyl((S)-1-(((S)-1-((4-amino-5-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(A2)

Protected aniline A1 (18 g, 34.19 mMol) was solubilised in a mixture ofMeOH and H₂O 10:1 (165 mL) and K₂CO₃ was added (10 g, 72.36 mMol). Themixture was stirred at 50° C. until complete. The mixture was vacceddown to almost dryness and the residue was taken up with CH₂Cl₂ andwashed with H₂O and brine, before being dried over MgSO₄, filtered andevaporated. The crude product was purified by silica gel chromatography(CHCl₃/MeOH, 100% to 7:3). The isolated product A2 was contaminated witha co-eluting impurity (10.71 g, 73% yield). LC/MS (Method B): ES⁺=1.46min, m/z 431.7 [M+H]⁺.

c)Allyl((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methylbutan-2-yl)carbamate(A4)

Aniline A2 (450 mg, 1.045 mMol), lactone A3 (280 mg, 1.064 mMol) andpyridinium p-toluenesulfonate (273 mg, 1.086 mMol) were solubilised intoluene (20 mL) and the mixture was heated to 130° C. (high reflux).Every now and then a few drops of MeOH is added to help solubilise themixture. After 7 h the crude reaction was vacced down to dryness. Thecrude product was purified by silica gel chromatography (CHCl₃/MeOH,100% to 95:5) to give product A4 (360 mg, 52.3% yield). LC/MS (MethodB): ES⁺=1.51 min, m/z 658.8 [M+H]⁺.

d)Allyl(S)-2-amino-N—((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide(A5)

Excess piperidine was added (642 μL) to a solution of A4 (543 mg, 0.82mMol) and PdP(Ph₃)₄ (89 mg, 0.08 mMol) in CH₂Cl₂ (15 mL). The mixturewas allowed to stir at room temperature for 20 min, at which point thereaction had gone to completion (as monitored by LC/MS). The reactionmixture was diluted with CH₂Cl₂ (25 mL) and the organic phase was washedwith H₂O (25 mL) and brine (25 mL). The organic phase was dried overMgSO₄, filtered and excess solvent removed by rotary evaporation underreduced pressure to afford crude product A5 which was used as such inthe next step. LC/MS (Method B): ES⁺=1.15 min, m/z 574.6 [M+H]⁺.

e)1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N—((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide(1)

Pyridine (83 μL, 1.03 mMol) and Mal-dPEG₈-OTFP (767 mg, 1.03 mMol) wereadded to a solution of crude A5 (assumed 1.03 mMol) in dry CH₂Cl₂ (50mL) under an argon atmosphere. The reaction was stirred overnight and asthe reaction was not complete 0.5 eq. of Mal-dPEG₈-OTFP was added to tryto push the reaction. The reaction was diluted with CH₂Cl₂ (25 mL) andthe organic phase was washed with H₂O (2×50 mL) and brine before beingdried over MgSO₄, filtered and excess solvent removed by rotaryevaporation under reduced pressure by rotary evaporation under reducedpressure. The crude was purified by reverse phase HPLC (gradient ofH₂O/CH₃CN+0.05% FA) and freezedried to give 1 (1.189 g, 31% yield over 2steps). LC/MS (Method B): ES⁺=1.43 min, m/z 1149.3 [M+H]⁺. LC/MS (MethodC): ES⁺=5.37 min, m/z 1149.4 [M+H]⁺.

Example 2

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N—((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)hexanamide(2)

Mal-caproic acid (56 mg, 0.26 mMol) and EDCI.HCl (51 mg, 0.26 mMol) wereadded to a solution of crude A5 (assumed 0.26 mMol) in dry CH₂Cl₂ (20mL) under an argon atmosphere. The reaction was stirred overnight and asthe reaction was incomplete, another 0.5 eq of Mal-caproic acid andEDCI.HCl were added. The reaction was diluted with CH₂Cl₂ (25 mL) andthe organic phase was washed with H₂O (2×50 mL) and brine before beingdried over MgSO₄, filtered and excess solvent removed by rotaryevaporation under reduced pressure by rotary evaporation under reducedpressure. The crude was purified by silica gel column chromatography(CHCl₃/MeOH 95:5) to give 2 (31.6 mg, 20% yield over 2 steps). LC/MS(Method B): ES⁺=1.56 min, m/z 767.8 [M+H]⁺. LC/MS (Method C) 15 min:ES⁺=6.05 min, m/z 767.8 [M+H]⁺.

Example 3

(S)-2-(2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)acetamido)-N—((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide(3)

Azido-dPEG₃-acid (77.5 mg, 0.31 mMol) and EDCI.HCl (60 mg, 0.31 mMol)were added to a solution of crude A5 (assumed 0.31 mMol) in dry CH₂Cl₂(20 mL) under an argon atmosphere. The reaction was stirred overnightand as the reaction was incomplete, another 0.5 eq. of azido-dPEG₃-OHand EDCI.HCl were added. The reaction was diluted with CH₂Cl₂ (25 mL)and the organic phase was washed with H₂O (2×50 mL) and brine beforebeing dried over MgSO₄, filtered and excess solvent removed by rotaryevaporation under reduced pressure by rotary evaporation under reducedpressure. The crude was purified by preparative HPLC and the fractionswere freezedried to give pure 3 (92.2 mg, 24.7% yield over 2 steps).LC/MS (Method B): ES⁺=1.69 min, m/z 789.9 [M+H]⁺. LC/MS (Method C):ES⁺=6.68 min, m/z 790.0 [M+H]⁺.

Example 4

N—((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-4,7,10,13,16-pentaoxanonadec-18-ynamide(4)

Propargyl-dPEG₅-acid (56 mg, 0.19 mMol) and EDCI.HCl (37 mg, 0.19 mMol)were added to a solution of crude A5 (assumed 0.19 mMol) in dry CH₂Cl₂(10 mL) under an argon atmosphere. The reaction was stirred overnightand as the reaction was incomplete, another 0.5 eq. ofPropargyl-dPEG₅-OH and EDCI.HCl were added. The reaction was dilutedwith CH₂CL₂ (25 mL) and the organic phase was washed with H₂O (2×50 mL)and brine before being dried over MgSO₄, filtered and excess solventremoved by rotary evaporation under reduced pressure by rotaryevaporation under reduced pressure. The crude was purified bypreparative HPLC and the fractions were freezedried to give pure 4 (22mg, 16.7% yield over 2 steps). LC/MS (Method B): ES⁺=1.54 min, m/z 860.9[M+H]⁺. LCMS (Method C): ES+=5.57 min, m/z 860.9 [M+H]⁺.

Example 5

(S)-2-(2-(4-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)phenyl)acetamido)-N—((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)-3-methylbutanamide(5)

PM-acetic-OSu (64 mg, 0.19 mMol) was added to a solution of crude A5(assumed 0.19 mMol) in dry CH₂Cl₂ (10 mL) under an argon atmosphere. Thereaction was not proceeding so DIPEA (51 μL, 0.28 mMol) was added. Thereaction was stirred until complete. The mixture was diluted with CH₂Cl₂(25 mL) and the organic phase was washed with H₂O (2×50 mL) and brinebefore being dried over MgSO₄, filtered and excess solvent removed byrotary evaporation under reduced pressure by rotary evaporation underreduced pressure. The crude was purified by preparative HPLC and thefractions were freezedried to give pure 5 (2.5 mg, 1.6% yield over 2steps). LC/MS (Method B): ES⁺=1.54 min, m/z 787.7 [M+H]⁺. LC/MS (MethodC): ES⁺=5.61 min, m/z 787.8 [M+H]⁺.

Example 6

(R)-2-((3-nitropyridin-2-yl)disulfaneyl)propyl((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)carbamate(6)

(i) (2R)-2-[(3-nitro-2-pyridyl)disulfanyl]propan-1-ol A6 (25 mg, 0.1015mmol, 1.0 eq.) was dissolved in dichloromethane (1 mL). Pyridine (8.5μL, 0.11 mmol, 1.0 eq.), then triphosgene (11 mg, 0.0370685 mmol, 0.33eq.) were added and the mixture stirred under Ar for 45 min, whereuponLCMS (Et₂NH quench) indicated the formation of the correspondingcarbamate.

(ii)(S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione(111) (43 mg, 0.09026 mmol, 1.0 eq.) was dissolved in dichloromethane (2mL), N,N-diisopropylethylamine (42 μL, 0.241 mmol, 2.7 eq.) and pyridine(25 μL, 0.309 mmol, 3.4 eq.). The reaction mixture from step (i) wasadded and the mixture stirred for 30 min, whereupon LCMS indicated thereaction was complete. The reaction mixture was concentrated in vacuoand purified by isolera chromatography (0-4% MeOH in CH₂O1₂) to afford 6(22 mg, 0.03256 mmol, 36% Yield, QC=96.8%) as a yellow solid. LC/MS(Method B): RT=1.86 min, 676.6 [M+H]⁺.

Example 7

6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-((2-(((S)-1-((2-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)hexanamide(7)

Compound I18 (259 mg, 0.3588 mmol) was solubilised in CH₂Cl₂ (25 mL).The starting material was not soluble at all so DMA (1 mL) was added. Asno improvement was observed, DIPEA (68 μL, 0.390 mmol) was added and allthe solid went in solution. Maleimide caproic acid (69 mg, 0.358 mmol)was added and the mixture left to stir at r.t. overnight and which pointLCMS analysis revealed the reaction to be complete. The reaction mixturewas quenched with MeOH (2 mL) and vacced down to dryness. The crudeproduct was purified by preparative HPLC and subsequently freezedried togive compound 7 as an ochre solid (38.2 mg, 11% yield). Analytical data:LCMS 3 min: ES⁺=1.47 min, m/z 916.2 [M+H]⁺ LCMS 15 min: ES⁺=5.46 min,m/z 916.1 [M+H]⁺.

Example 8

1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-(2-((2-(((S)-1-((2-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-2-oxoethyl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)-3,6,9,12,15,18,21,24-octaoxaheptacosan-27-amide(8)

Compound I18 (70 mg, 0.096 mmol) was solubilised in CH₂Cl₂ (5 mL). Thestarting material was not soluble at all so DMA (0.5 mL) was added. Asno improvement was observed, DIPEA (19 μL, 0.106 mmol) was added and allthe solid went in solution. Mal-dPEG₈-OH (63 mg, 0.106 mmol) andEDCI.HCl (19 mg, 0.099 mMol) were added and the mixture left to stir atr.t. overnight and which point LCMS analysis revealed the reaction to becomplete. The reaction mixture was quenched with MeOH (2 mL) and vacceddown to dryness. The crude product was purified by preparative HPLC andsubsequently freezedried to give 8 as an ochre solid (30 mg, 24% yield).LCMS 3 min: ES⁺=1.44 min, m/z 1297.6 [M+H]⁺.

Example 9—Alternate Synthesis of 1

(S)-4-amino-9-ethyl-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinoline-10,13-dione 111(371 mg, 0.779 mmol, 1.0 eq.) was dissolved in dichloromethane (30 mL).N,N-diisopropylethylamine (69 μL, 0.396 mmol, 0.51 eq.), and(2S)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-3-methyl-butanoyl]amino]propanoicacid (664 mg, 0.871 mmol, 1.1 eq.) in N,N-dimethylacetamide (10 mL) wereadded, followed by EDCI.HCl (226 mg, 1.18 mmol, 1.5 eq.) and the mixturestirred for 2 h, whereupon LCMS indicated good conversion, but that thereaction had stalled. The reaction mixture was warmed to 30° C. andstirred for 30 min, LCMS indicated no change so CH₂Cl₂ was removed invacuo and Et₂O added to the resulting DMA solution. The precipitated oilwas collected, Et₂O removed in vacuo and the precipitation processrepeated. The combined precipitates were purified by HPLC (10-60% B in Aover 13 min) to afford 1 (200 mg, 0.174 mmol, 98% purity, 22% Yield) asa yellow residue after freeze-drying. LC/MS (method A): retention time1.44 mins (ES⁺) m/z 1149 [M+H]⁺

¹H NMR (600 MHz, Chloroform-d) δ 8.81 (s, 1H), 7.83 (s, 2H), 7.48 (s,1H), 7.18 (dd, J=18.7, 7.5 Hz, 2H), 6.69 (s, 2H), 6.43 (s, 1H), 5.68 (d,J=16.1 Hz, 1H), 5.27 (d, J=16.1 Hz, 1H), 5.03 (d, J=18.4 Hz, 1H), 4.90(d, J=18.4 Hz, 1H), 4.75 (p, J=7.2 Hz, 1H), 4.32 (dd, J=7.4, 5.8 Hz,1H), 4.05 (s, 1H), 3.83 (t, J=7.2 Hz, 3H), 3.78-3.68 (m, 3H), 3.68-3.57(m, 31H), 3.53 (t, J=5.1 Hz, 3H), 3.40 (q, J=5.3 Hz, 2H), 3.06-2.91 (m,3H), 2.84 (dt, J=16.3, 6.2 Hz, 1H), 2.63 (ddd, J=14.8, 8.5, 4.2 Hz, 1H),2.57-2.44 (m, 4H), 2.30 (dq, J=13.4, 6.7 Hz, 1H), 2.10 (p, J=6.4 Hz,3H), 1.91 (ddt, J=16.8, 14.3, 7.2 Hz, 3H), 1.54 (d, J=7.1 Hz, 3H), 1.02(dd, J=15.5, 6.9 Hz, 10H).

Example 10—Alternate Synthesis of A2

Allyl((S)-1-(((S)-1-((4-amino-5-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate(A2)

EDCI.HCl (7.71 g, 31.2 mMol) was added to a solution of alloc-Val-Ala-OH(8.49 g, 31.2 mmol) in CH₂Cl₂ (200 mL) and stirred for 15 min or untilsolubilised. I16 (5 g, 28.3 mMol) was subsequently added and theresulting mixture was left to stir until the reaction was completed. Thevolatiles were removed under reduced pressure. The crude product wastaken up in Et₂O (50 mL) and the mixture sonicated for 3 min. The solidwas filtrated and taken up again in CH2Cl2 (50 mL), sonicated for 3 minand filtered again to give pure product A2 as a grey solid (12.21 g, 79%yield). LC/MS (Method B): ES⁺=1.47 min, m/z 431.5 [M+H]⁺.

Example 11

a) (9H-fluoren-9-yl)methylN2-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oyl)-N5-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-L-glutaminate(A7)

EDCI.HCl (0.10 mmol, 1.2 eq) was added to a solution of A5 (0.087 mmol,1.0 eq) and Mal-PEG₈-Glu-OH (0.10 mmol, 1.2 eq) in DCM (5 mL) and theresulting mixture stirred at room temperature overnight. The reactionmixture was evaporated to dryness and purified by column (8-12%MeOH/DCM) to leave the product as a white solid. Yield=80 mg (63%).LC/MS (Method B) rt 1.66 min m/z (1456.2) M+H.

b)N₂-(1-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3-oxo-7,10,13,16,19,22,25,28-octaoxa-4-azahentriacontan-31-oyl)-N5-((S)-1-(((S)-1-(((S)-9-ethyl-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-4-yl)amino)-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)-L-glutamine(9)

1-Methylpyrrolidine (200 μL) was added to a solution of A7 (0.06 mmol)in DMF (0.8 mL) and stirred at room temperature for 10 mins. The solventwas removed under vacuum and the residue purified by prep HPLC (30%MeCN/water+0.05% formic acid over 8.5 mins). Fractions containingproduct were freeze dried to give the product as an off-white solid.Yield=23 mg (30%). LC/MS (Method B) rt 1.43 min m/z (1278.4) M+H.

Example 12—Conjugation

Herceptin-C239i Antibody

Herceptin antibodies were engineered to have cysteine inserted betweenthe 239 and 240 positions were produced following the methods describedin Dimasi, N., et al., Molecular Pharmaceutics, 2017, 14, 1501-1516(DOI: 5 10.1021/acs.molpharmaceut.6b00995).

ConjA

A 50 mM solution of DL-dithiothreitol (DTT) in phosphate-buffered salinepH 7.4 (PBS) was added (150 molar equivalent/antibody, 40 micromoles,800 μL) to a 10 mL solution of Herceptin-C239i antibody (40 mg, 267nanomoles) in reduction buffer containing PBS and 1 mMethylenediaminetetraacetic acid (EDTA) and a final antibodyconcentration of 4.0 mg/mL. The reduction mixture was allowed to reactat room temperature for 4 hours 45 minutes (or until full reduction isobserved by UHPLC) in an orbital shaker with gentle (60 rpm) shaking.The reduced antibody was buffer exchanged, via spin filtercentrifugation, into a reoxidation buffer containing PBS and 1 mM EDTAto remove all the excess reducing agent. A 50 mM solution ofdehydroascorbic acid (DHAA, 20 molar equivalent/antibody, 5.33micromoles, 106.7 μL) in DMSO was added and the reoxidation mixture wasallowed to react for 16 hours at room temperature with gentle (60 rpm)shaking at an antibody concentration of 4 mg/mL (or more DHAA added andreaction left for longer until full reoxidation of the cysteine thiolsto reform the inter-chain cysteine disulfides is observed by UHPLC). Thereoxidation mixture was then sterile-filtered and diluted in aconjugation buffer containing PBS and 1 mM EDTA for a final antibodyconcentration of 3.6 mg/mL. Compound 1 was added as a DMSO solution (10molar equivalent/antibody, 1.33 micromoles, in 0.55 mL DMSO) to 5.0 mLof this reoxidised antibody solution (20 mg, 133 nanomoles) fora 10%(v/v) final DMSO concentration. The solution was mixed for 2 hours atroom temperature, then the conjugation was quenched by addition ofN-acetyl cysteine (6.67 micromoles, 67 μL at 100 mM), then purified byspin filtration into PBS using a 15 mL Amicon Ultracell 30 kDa MWCO spinfilter, sterile-filtered and analysed. UHPLC analysis on a ShimadzuProminence system using a Thermo Scientific MAbPac 50 mm×2.1 mm columneluting with a gradient of water and acetonitrile on a reduced sample ofConjA at 214 nm and 330 nm (Compound 1 specific) shows unconjugatedlight chains and a mixture of unconjugated heavy chains and heavy chainsattached to a single molecule of Compound 1, consistent with adrug-per-antibody ratio (DAR) of 1.89 molecules of Compound 1 perantibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of ConjA at 280 nm shows a monomerpurity of 98%. UHPLC SEC analysis gives a concentration of final ConjAat 2.14 mg/mL in 6.5 mL, obtained mass of ConjA is 13.9 mg (70% yield).

ConjA*

A 10 mM solution of Tris(2-carboxyethyl)phosphine (TCEP) inphosphate-buffered saline pH 7.4 (PBS) was added (10 molarequivalent/antibody, 400 nanomoles, 40 μL) to a 2.4 mL solution ofTratuzumab antibody (6 mg, 40 nanomoles) in reduction buffer containingPBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and a final antibodyconcentration of 2.5 mg/mL. The reduction mixture was allowed to reactat room temperature for 16 hours (or until full reduction is observed byUHPLC) in an orbital shaker with gentle (60 rpm) shaking. The reducedantibody solution was buffer exchanged (to remove all the excessreducing agent), via spin filter centrifugation, into a conjugationbuffer containing PBS and 1 mM EDTA for a final antibody concentrationof 2.0 mg/mL. Compound 1 was added as a DMSO solution (20 molarequivalent/antibody, 400 nanomoles, in 0.15 mL DMSO) to 1.35 mL of thisreduced antibody solution (3 mg, 20 nanomoles) for a 10% (v/v) finalDMSO concentration. The solution was mixed for 2 hours at roomtemperature, then the conjugation was quenched by addition of N-acetylcysteine (2 micromoles, 20 μL at 100 mM), then purified via spin filtercentrifugation using a 15 mL Amicon Ultracell 30KDa MWCO spin filter,sterile-filtered and analysed.

UHPLC analysis on a Shimadzu Prominence system using a Thermo ScientificMAbPac 50 mm×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of ConjA* at 214 nm and 330 nm(Compound 1 specific) shows a mixture of unconjugated light chains,light chains attached to a single molecule of Compound 1, unconjugatedheavy chains and heavy chains attached to up to three molecules ofCompound 1, consistent with a drug-per-antibody ratio (DAR) of 7.89molecules of Compound 1 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of ConjA* at 280 nm shows amonomer purity of 98.5%. UHPLC SEC analysis gives a concentration offinal ConjA* at 2.02 mg/mL in 1.25 mL, obtained mass of ConjA* is 2.5 mg(84% yield).

ConjB

A 10 mM solution of Tris(2-carboxyethyl)phosphine (TCEP) inphosphate-buffered saline pH 7.4 (PBS) was added (10 molarequivalent/antibody, 3.56 micromoles, 356 μL) to a 11.1 mL solution ofTratuzumab antibody (53.4 mg, 356 nanomoles) in reduction buffercontaining PBS, pH 7.4 and 1 mM ethylenediaminetetraacetic acid (EDTA)and a final antibody concentration of 4.84 mg/mL. The reduction mixturewas allowed to react at 37° C. for 1 hour 30 minutes (or until fullreduction is observed by UHPLC) in an orbital shaker with gentle (60rpm) shaking. Compound 2 was added as a DMSO solution (15 molarequivalent/antibody, 5.1 micromoles, in 1.2 mL DMSO) to 10.5 mL of thisreduced antibody solution (50.8 mg, 339 nanomoles) for a 10% (v/v) finalDMSO concentration. The solution was mixed for 1 hour 30 minutes at roomtemperature, then the conjugation was quenched by addition of N-acetylcysteine (25.4 micromoles, 254 μL at 100 mM), then purified on an AKTA™Start FPLC using a GE Healthcare HiLoad™ 26/600 column packed withSuperdex 200 PG, eluting with 2.6 mL/min PBS. Fractions corresponding toConjB monomer peak were pooled, concentrated and buffer exchanged into25 mM Histidine 205 mM Sucrose pH 6.0 buffer using a 15 mL AmiconUltracell 50KDa MWCO spin filter, sterile-filtered and analysed.

UHPLC analysis on a Shimadzu Prominence system using a Thermo ScientificMAbPac 50 mm×2.1 mm column eluting with a gradient of water andacetonitrile on a reduced sample of ConjB at 214 nm and 330 nm (Compound2 specific) shows a mixture of unconjugated light chains, light chainsattached to a single molecule of Compound 2, unconjugated heavy chainsand heavy chains attached to up to three molecules of Compound 2,consistent with a drug-per-antibody ratio (DAR) of 7.93 molecules ofCompound 2 per antibody. UHPLC analysis on a Shimadzu Prominence systemusing a Tosoh Bioscience TSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column(with a 4 μm 3.0×20 mm guard column) eluting with 0.3 mL/minutesterile-filtered SEC buffer containing 200 mM potassium phosphate pH6.95, 250 mM potassium chloride and 10% isopropanol (v/v) on a sample ofConjB at 280 nm shows a monomer purity of 98.9%. UHPLC SEC analysisgives a concentration of final ConjB at 2.4 mg/mL in 16 mL, obtainedmass of ConjB is 38.4 mg (84% yield).

ConjC

A 10 mM solution of Tris(2-carboxyethyl)phosphine (TCEP) inphosphate-buffered saline pH 7.4 (PBS) was added (40 molarequivalent/antibody, 11.2 micromoles, 1.12 mL) to a 20 mL solution ofHerceptin-C239i antibody (42 mg, 280 nanomoles) in reduction buffercontaining PBS and 1 mM ethylenediaminetetraacetic acid (EDTA) and afinal antibody concentration of 2.1 mg/mL. The reduction mixture wasallowed to react at room temperature for 16 hours (or until fullreduction is observed by UHPLC) in an orbital shaker with gentle (60rpm) shaking. The reduced antibody was buffer exchanged, via spin filtercentrifugation, into a reoxidation buffer containing PBS and 1 mM EDTAto remove all the excess reducing agent. A 50 mM solution ofdehydroascorbic acid (DHAA, 30 molar equivalent/antibody, 7.0micromoles, 141 μL) in DMSO was added to 22 mL of this reduced bufferexchanged antibody (35.2 mg, 235 nanomoles) and the reoxidation mixturewas allowed to react for 2 hours and 30 minutes at room temperature withgentle (60 rpm) shaking at an antibody concentration of 1.6 mg/mL (ormore DHAA added and reaction left for longer until full reoxidation ofthe cysteine thiols to reform the inter-chain cysteine disulfides isobserved by UHPLC). The reoxidation mixture was then sterile-filtered.Compound 6 was added as a DMSO solution (20 molar equivalent/antibody,2.3 micromoles, in 1.36 mL DMSO) to 11.0 mL of this reoxidised antibodysolution (17.6 mg, 117 nanomoles) pH adjusted with 1.22 mL of 1 M SodiumBicarbonate for a 10% (v/v) final DMSO concentration and 10% (v/v) 1 Msodium bicarbonate. The solution was left to react at room temperaturefor 2 hours with gentle shaking. Then the conjugation was quenched byaddition of N-acetyl cysteine (12 micromoles, 117 μL at 100 mM), thenpurified and buffer exchanged into 25 mM Histidine 205 mM Sucrose pH 6.0buffer using a 15 mL Amicon Ultracell 50 kDa MWCO spin filter,sterile-filtered and analysed.

UHPLC analysis on a Shimadzu Prominence system using a Sepax ProteomixHIC Butyl-NP5 4.6×35 mm 5 μm column eluting with a gradient of 25 mMsodium phosphate, 1.5 M ammonium sulphate pH 7.4 buffer and 20%acetonitrile (v/v) in 25 mM sodium phosphate pH 7.4 buffer on intactsample of ConjC at 214 nm and 330 nm (Compound 6 specific) showedunconjugated and conjugated antibody attached to one or two molecules ofCompound 6, consistent with a drug-per-antibody ratio (DAR) of 1.42molecules of Compound 6 per antibody.

UHPLC analysis on a Shimadzu Prominence system using a Tosoh BioscienceTSKgel SuperSW mAb HTP 4 μm 4.6×150 mm column (with a 4 μm 3.0×20 mmguard column) eluting with 0.3 mL/minute sterile-filtered SEC buffercontaining 200 mM potassium phosphate pH 6.95, 250 mM potassium chlorideand 10% isopropanol (v/v) on a sample of ConjC at 280 nm shows a monomerpurity of 98%. UHPLC SEC analysis gives a concentration of final ConjCat 1.06 mg/mL in 10.1 mL, obtained mass of ConjC is 10.7 mg (61% yield).

Example 13—In Vitro Assay

Solid test material was dissolved in DMSO to a 2 mM stock solution, fromwhich eight serial dilutions were made at a 1:10 ratio in DMSO andstored at −20° C. until use.

Adherent NCI-N87 cells were washed with D-PBS and detached withTrypsin-EDTA, cell density and viability were then determined induplicate by Trypan blue exclusion assay using an automated cell counter(LUNA-II™). Cell suspension was diluted to 1×10⁵ cells/ml in growthmedia (RPMI 1640 with Glutamax+10% (v/v) HyClone™ Fetal Bovine Serum)and vortexed before dispensing 2 mL per well into sterile 3 mLpolypropylene plates. Warhead dilutions were then dispensed into theappropriate wells at 10 μl/well and mixed by repeat pipetting. Forcontrol wells 10 μl of DMSO was dispensed onto 2 mL cell suspension, andthoroughly mixed. 100 μl of each sample was then aliquoted into 2replicate wells of a sterile flat 96-well microplate and incubated in a37° C. CO₂-gassed (5%) incubator. At the end of the incubation periodtime (7 days), cell viability was measured by CellTiter 96™ aqueous One(MTS) assay, which was dispensed at 20 μl/well and incubated for 4 hoursat 37° C., 5% CO₂. Plates were then read on an EnVision™ Multi-labelPlate Reader (Perkin Elmer) using absorbance at 490 nm.

Cell survival percentage was calculated from the mean absorbance of the2 replicate wells for each sample, compared to the mean absorbance inthe two control wells treated with DMSO only (100%). The IC₅₀ wasdetermined by fitting each data set to sigmoidal dose-response curveswith a variable slope using the non-linear curve fit algorithm on theGraphPad Prism software (San Diego, Calif.).

All the experiments in this report were carried out and tested in threeindependent experiments. Data are reported as the mean of the threeindependent replicates.

IC₅₀ (nM) I11 0.3854

Example 14—ADC In Vitro Assay

The concentration and viability of cells from a sub-confluent (80-90%confluency) T75 flask are measured by trypan blue staining, and countedusing the LUNA-II™ Automated Cell Counter. Cells were diluted to2×10⁵/ml, dispensed (50 μl per well) into 96-well flat-bottom plates.

A stock solution (1 ml) of antibody drug conjugate (ADC) (20 μg/ml) wasmade by dilution of filter-sterilised ADC into cell culture medium. Aset of 8×10-fold dilutions of stock ADC were made in a 24-well plate byserial transfer of 100 μl into 900 μl of cell culture medium. ADCdilution was dispensed (50 μl per well) into 4 replicate wells of the96-well plate, containing 50 μl cell suspension seeded the previously.Control wells received 50 μl cell culture medium. The 96-well platecontaining cells and ADCs was incubated at 37° C. in a CO₂-gassedincubator for the exposure time.

At the end of the incubation period, cell viability was measured by MTSassay. MTS (Promega) was dispensed (20 μl per well) into each well andincubated for 4 hours at 37° C. in the CO₂-gassed incubator. Wellabsorbance was measured at 490 nm. Percentage cell survival wascalculated from the mean absorbance in the 4 ADC-treated wells comparedto the mean absorbance in the 4 control untreated wells (100%). IC₅₀ wasdetermined from the dose-response data using GraphPad Prism using thenon-linear curve fit algorithm: sigmoidal dose-response curve withvariable slope.

ADC incubation times were 4 days with MDA-MB-468 and 7 days for NCI-N87.MDA-MB-468 and NCI-N87 were cultured in RPMI 1640 with Glutamax+10%(v/v) HyClone™ Fetal Bovine Serum. NCI-N87 is a Her2-expressing cellline and MDA-MB-468 is a Her2 negative cell line.

EC₅₀ (μg/ml) NCI-N87 MDA-MB-468 ConjA 0.1176 >10 ConjA* 0.01634 >10ConjB 0.01857 >10 ConjC 0.1452 >10

Example 15—ADC In Vivo Assay

Methods and Materials

Mice

Female severe combined immunodeficient mice (Fox Chase SCID™,CB17/Icr-Prkdc^(scid)/IcoIcrCrl, Charles River) were eight weeks oldwith a body weight (BW) range of 14.5 to 20.0 grams on Day 1 of thestudy. The animals were fed ad libitum water (reverse osmosis, 1 ppmCl), and NIH 31 Modified and Irradiated Lab Diet™ consisting of 18.0%crude protein, 5.0% crude fat, and 5.0% crude fiber. The mice werehoused on irradiated Enrich-o'cobs™ Laboratory Animal Bedding in staticmicroisolators on a 12-hour light cycle at 20-22° C. and 40-60%humidity. CR Discovery Services specifically complies with therecommendations of the Guide for Care and Use of Laboratory Animals withrespect to restraint, husbandry, surgical procedures, feed and fluidregulation, and veterinary care. The animal care and use program at CRDiscovery Services is accredited by the Association for Assessment andAccreditation of Laboratory Animal Care International (AAALAC), whichassures compliance with accepted standards for the care and use oflaboratory animals.

Tumor Cell Culture

Human NCI-N87 gastric carcinoma lymphoma cells were cultured inRPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mMglutamine, 100 units/mL penicillin, 100 μg/mL streptomycin sulfate and25 μg/mL gentamicin. The cells were grown in tissue culture flasks in ahumidified incubator at 37° C., in an atmosphere of 5% CO₂ and 95% air.

In Vivo Implantation and Tumor Growth

The NCI-N87 cells used for implantation were harvested during log phasegrowth and resuspended in phosphate buffered saline (PBS) containing 50%Matrigel™ (BD Biosciences). On the day of tumor implant, each test mousewas injected subcutaneously in the right flank with 1×10⁷ cells (0.1 mLcell suspension), and tumor growth was monitored as the average sizeapproached the target range of 100 to 150 mm³. Twelve days later,designated as Day 1 of the study, mice were sorted according tocalculated tumor size into fourteen groups, seven designated forefficacy evaluation (n=10) and seven designated for sample collection(n=3) each consisting of animals with individual tumor volumes rangingfrom 108 to 172 mm³ and group mean tumor volumes of 120-124 mm³. Tumorswere measured in two dimensions using calipers, and volume wascalculated using the formula:

Tumor Volume (mm³)=(w ² ×l)/2

where w=width and l=length, in mm, of the tumor. Tumor weight may beestimated with the assumption that 1 mg is equivalent to 1 mm³ of tumorvolume.

Therapeutic Agents

ConjA* was stored protected from light at 4° C. Sterile PBS was used todose the vehicle control group.

Treatment

On Day 1 of the study, female SCID mice bearing established NCI-N87xenografts were sorted into groups. An aliquot of stock solution wasdiluted with PBS to the appropriate concentration. The agent wasadministered i.v. via tail vein injection once on Day 1. The dosingvolume was 0.2 mL per 20 grams of body weight (10 mL/kg), and was scaledto the body weight of each individual animal.

Group 1 mice received PBS vehicle, and served as the control group.Group 2 received ConjA* at 4 mg/kg.

Tumors were measured using calipers twice per week, and each animal waseuthanized when its tumor reached the endpoint volume of 800 mm³ or atthe end of the study (Day 68), whichever came first. Animals that exitedthe study for tumor volume endpoint were documented as euthanized fortumor progression (TP), with the date of euthanasia.

Criteria for Regression Responses

Treatment efficacy may be determined from the incidence and magnitude ofregression responses observed during the study. Treatment may causepartial regression (PR) or complete regression (CR) of the tumor in ananimal. In a PR response, the tumor volume was 50% or less of its Day 1volume for three consecutive measurements during the course of thestudy, and equal to or greater than 13.5 mm³ for one or more of thesethree measurements. In a CR response, the tumor volume was less than13.5 mm³ for three consecutive measurements during the course of thestudy. Animals were scored only once during the study for a PR or CRevent and only as CR if both PR and CR criteria were satisfied. Ananimal with a CR response at the termination of a study was additionallyclassified as a tumor-free survivor (TFS). Animals were monitored forregression responses.

Toxicity

Animals were weighed daily on Days 1-5, then twice per week until thecompletion of the study. The mice were observed frequently for overtsigns of any adverse, treatment-related (TR) side effects, and clinicalsigns were recorded when observed. Individual body weight was monitoredas per protocol, and any animal with weight loss exceeding 30% for onemeasurement or exceeding 25% for three consecutive measurements waseuthanized as a TR death. Group mean body weight loss was also monitoredaccording to CR Discovery Services protocol. Acceptable toxicity wasdefined as a group mean body weight (BW) loss of less than 20% duringthe study and no more than 10% TR deaths.

Results

FIG. 1 presents plots of mean tumor growth in which:

Vehicle ● ConjA* ♦

Group 1 mice received PBS vehicle i.v. qd×1 and served as the controlgroup. The median TTE for Group 1 was 24.8 days. All control tumorsattained the 800 mm³ endpoint.

Group 2 received ConjA* at 4 mg/kg i.v. qd×1. CRs were observed in all10 mice which were additionally classified as TFSs at the termination ofthe study.

In the treatment group the body weight nadir was −9.5% on day 50 of thestudy. No TR deaths were observed.

STATEMENTS OF INVENTION

1. A compound with the formula I:

and salts and solvates thereof, wherein R^(L) is a linker for connectionto a Ligand Unit, which is selected from:

-   -   (ia):

-   -   wherein    -   Q is:

where Q^(X) is such that Q is an amino-acid residue, a dipeptideresidue, a tripeptide residue or a tetrapeptide residue; X is:

-   -   where a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1,        d=0 to 5, wherein at least b1 or b2=0 and at least c1 or c2=0;    -   G^(L) is a linker for connecting to a Ligand Unit;    -   (ib):

-   -   where R^(L1) and R^(L2) are independently selected from H and        methyl, or together with the carbon atom to which they are bound        form a cyclopropylene or cyclobutylene group; and    -   e is 0 or 1.

2. The compound according to statement 1, wherein R^(L) is of formulaIa.

3. The compound according to statement 2, wherein Q is an amino acidresidue.

4. The compound according to statement 3, wherein Q is selected from:Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp.

5. The compound according to statement 2, wherein Q is a dipeptideresidue.

6. The compound according to statement 5, wherein Q is selected from:

-   -   ^(NH)-Phe-Lys-^(C═O),    -   ^(NH)-Val-Ala-^(C═O),    -   ^(NH)-Val-Lys-^(C═O),    -   ^(NH)-Ala-Lys-^(C═O),    -   ^(NH)-Val-Cit-^(C═O),    -   ^(NH)-Phe-Cit-^(C═O),    -   ^(NH)-Leu-Cit-^(C═O),    -   ^(NH)-Ile-Cit-^(C═O),    -   ^(NH)-Phe-Arg-^(C═O),    -   ^(NH)-Trp-Cit-^(C═O), and    -   ^(NH)-Gly-Val-^(C═O).

7. The compound according to statement 6, wherein Q is selected from^(NH)-Phe-Lys-^(C═O), ^(NH)-Val-Cit-^(C═O) and ^(NH)-Val-Ala-^(C═O).

8. The compound according to statement 2, wherein Q is a tripeptideresidue.

9. The compound according to statement 8, wherein Q is selected from:

-   -   ^(NH)-Val-Ala-^(C═O),    -   ^(NH)-Val-Cit-^(C═O),    -   ^(NH)-αVal-Ala-^(C═O), and    -   ^(NH)-αVal-Cit-^(C═O).

10. The compound according to statement 2, wherein Q is a tetrapeptideresidue.

11. The compound according to statement 10, wherein Q is selected from:

-   -   ^(NH)-Gly-Gly-Phe-Gly^(C═O); and    -   ^(NH)-Gly-Phe-Gly-Gly^(C═O).

12. The compound according to statement 11, wherein Q is:

-   -   ^(NH)-Gly-Gly-Phe-Gly^(C═O).

13. The compound according to any one of statements 2 to 12, wherein ais 0 to 3.

14. The compound according to statement 13, wherein a is 0 or 1.

15. The compound according to statement 13, wherein a is 0.

16. The compound according to any one of statements 2 to 15, wherein b1is 0 to 8.

17. The compound according to statement 16, wherein b1 is 0.

18. The compound according to statement 16, wherein b1 is 2.

19. The compound according to statement 16, wherein b1 is 3.

20. The compound according to statement 16, wherein b1 is 4.

21. The compound according to statement 16, wherein b1 is 5.

22. The compound according to statement 16, wherein b1 is 8.

23. The compound according to any one of statements 2 to 15 and 17,wherein b2 is 0 to 8.

24. The compound according to statement 23, wherein b2 is 0.

25. The compound according to statement 23, wherein b2 is 2.

26. The compound according to statement 23, wherein b2 is 3.

27. The compound according to statement 23, wherein b2 is 4.

28. The compound according to statement 23, wherein b2 is 5.

29. The compound according to statement 23, wherein b2 is 8.

30. The compound according to any one of statements 2 to 29, wherein c1is 0.

31. The compound according to any one of statements 2 to 29, wherein c1is 1.

32. The compound according to any one of statements 2 to 31, wherein c2is 0.

33. The compound according to any one of statements 2 to 30, wherein c2is 1.

34. The compound according to any one of statements 2 to 33, wherein dis 0 to 3.

35. The compound according to statement 34, wherein d is 1 or 2.

36. The compound according to statement 34, wherein d is 2.

37. The compound according to any one of statements 2 to 33, wherein dis 5.

38. The compound according to any one of statements 2 to 12, wherein ais 0, b1 is 0, c1 is 1, c2 is 0 and d is 2, and b2 is from 0 to 8.

39. The compound according to statement 38, wherein b2 is 0, 2, 3, 4, 5or 8.

40. The compound according to any one of statements 2 to 12, wherein ais 1, b2 is 0, c1 is 0, c2 is 0 and d is 0, and b1 is from 0 to 8.

41. The compound according to statement 40, wherein b1 is 0, 2, 3, 4, 5or 8.

42. The compound according to any one of statements 2 to 12, wherein ais 0, b1 is 0, c1 is 0, c2 is 0 and d is 1, and b2 is from 0 to 8.

43. The compound according to statement 42, wherein b2 is 0, 2, 3, 4, 5or 8.

44. The compound according to any one of statements 2 to 12, wherein b1is 0, b2 is 0, c1 is 0, c2 is 0, one of a and d is 0, and the other of aand d is from 1 to 5.

45. The compound according to statement 44, wherein the other of a and dis 1 or 5.

46. The compound according to any one of statements 2 to 12, wherein ais 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 is from 0 to 8.

47. The compound according to statement 46, wherein b1 is 0, 2, 3, 4, 5or 8.

48. The compound according to any one of statements 2 to 47, whereinG^(L) is selected from

where Ar represents a C₅₋₆ arylene group, and X represents C₁₋₄ alkyl.

49. A compound according to statement 48, wherein G^(L) is selected fromG^(L1-1) and G^(L1-2).

50. A compound according to statement 48, wherein G^(L) is G^(L1-1).

51. The compound according to statement 1, wherein R^(L) is of formulaIb.

52. The compound according to statement 51, wherein both R^(L1) andR^(L2) are H.

53. The compound according to statement 51, wherein R^(L1) is H andR^(L2) is methyl.

54. The compound according to statement 51, wherein both R^(L1) andR^(L2) are methyl.

55. The compound according to statement 51, wherein R^(L1) and R^(L2)together with the carbon atom to which they are bound form acyclopropylene group.

56. The compound according to statement 51, wherein R^(L1) and R^(L2)together with the carbon atom to which they are bound form acyclobutylene group.

57. The compound according to any one of statements 51 to 56, wherein eis 0.

58. The compound according to any one of statements 51 to 56, wherein eis 1.

59. A conjugate of formula IV:

L-(D^(L))_(p)  (IV)

or a pharmaceutically acceptable salt or solvate thereof, wherein L is aLigand unit (i.e., a targeting agent), D^(L) is a Drug Linker unit thatis of formula III:

R^(LL) is a linker connected to the Ligand unit selected from

(ia′):

where Q and X are as defined in any one of statements 1 to 47 and G^(LL)is a linker connected to a Ligand Unit; and

(ib′):

where R^(L1) and R^(L2) are as defined in any one of statements 1 and 52to 56; and

p is an integer of from 1 to 20.

60. The conjugate according to statement 59, wherein G^(LL) is selectedfrom:

where Ar represents a C₅₋₆ arylene group and X represents C₁₋₄ alkyl.

61. The conjugate according to statement 60, wherein G^(LL) is selectedfrom G^(LL1-1) and G^(LL1-2).

62. The conjugate according to statement 61, wherein G^(LL) isG^(LL1-1).

63. The conjugate according to any one of statements 59 to 62, whereinthe Ligand Unit is a Cell Binding Agent.

64. The conjugate according to any one of statements 59 to 62, whereinthe Ligand Unit is an antibody or an active fragment thereof.

65. The conjugate according to statement 64, wherein the antibody orantibody fragment is an antibody or antibody fragment for atumour-associated antigen.

66. The conjugate according to statement 65, wherein the antibody orantibody fragment is an antibody which binds to one or moretumor-associated antigens or cell-surface receptors selected from(1)-(89):

(1) BMPR1B;

(2) E16;

(3) STEAP1;

(4) 0772P;

(5) MPF;

(6) Napi3b;

(7) Sema 5b;

(8) PSCA hlg;

(9) ETBR;

(10) MSG783;

(11) STEAP2;

(12) TrpM4;

(13) CRIPTO;

(14) CD21;

(15) CD79b;

(16) FcRH2;

(17) HER2;

(18) NCA;

(19) MDP;

(20) IL20R-alpha;

(21) Brevican;

(22) EphB2R;

(23) ASLG659;

(24) PSCA;

(25) GEDA;

(26) BAFF-R;

(27) CD22;

(28) CD79a;

(29) CXCR5;

(30) HLA-DOB;

(31) P2X5;

(32) CD72;

(33) LY64;

(34) FcRH1;

(35) IRTA2;

(36) TENB2;

(37) PSMA—FOLH1;

(38) SST;

(38.1) SSTR2;

(38.2) SSTR5;

(38.3) SSTR1;

(38.4) SSTR3;

(38.5) SSTR4;

(39) ITGAV;

(40) ITGB6;

(41) CEACAM5;

(42) MET;

(43) MUC1;

(44) CA9;

(45) EGFRvIII;

(46) CD33;

(47) CD19;

(48) IL2RA;

(49) AXL;

(50) CD30—TNFRSF8;

(51) BCMA—TNFRSF17;

(52) CT Ags—CTA;

(53) CD174 (Lewis Y)—FUT3;

(54) CLEC14A;

(55) GRP78—HSPA5;

(56) CD70;

(57) Stem Cell specific antigens;

(58) ASG-5;

(59) ENPP3;

(60) PRR4;

(61) GCC—GUCY2C;

(62) Liv-1—SLC39A6;

(63) 5T4;

(64) CD56—NCMA1;

(65) CanAg;

(66) FOLR1;

(67) GPNMB;

(68) TIM-1—HAVCR1;

(69) RG-1/Prostate tumor target Mindin—Mindin/RG-1;

(70) B7-H4—VTCN1;

(71) PTK7;

(72) CD37;

(73) CD138—SDC1;

(74) CD74;

(75) Claudins—CLs;

(76) EGFR;

(77) Her3;

(78) RON—MST1R;

(79) EPHA2;

(80) CD20—MS4A1;

(81) Tenascin C—TNC;

(82) FAP;

(83) DKK-1;

(84) CD52;

(85) CS1—SLAMF7;

(86) Endoglin—ENG;

(87) Annexin A1—ANXA1;

(88) V-CAM (CD106)—VCAM1;

(89) ASCT2 (SLC1A5).

67. The conjugate according to any one of statements 64 to 66, whereinthe antibody or antibody fragment is a cysteine-engineered antibody.

684. The conjugate according to any one of statements 64 to 67, whereinthe drug loading (p) of drugs (D) to antibody (Ab) is an integer from 1to about 10.

69. The conjugate according to statement 68, wherein p is 1, 2, 3, 4, 5,6, 7, 8, 9 or 10.

70. A mixture of conjugates according to any one of statements 64 to 69,wherein the average drug loading per antibody in the mixture ofantibody-drug conjugates is about 1 to about 10.

71. The conjugate or mixture according to any one of statements 59 to70, for use in therapy.

72. A pharmaceutical composition comprising the conjugate or mixture ofany one of statements 59 to 70 and a pharmaceutically acceptablediluent, carrier or excipient.

73. The conjugate or mixture according to any one of statements 59 to70, or the pharmaceutical composition according to statement 72, for usein the treatment of a proliferative disease in a subject.

74. The conjugate, mixture or pharmaceutical composition according tostatement 73, wherein the disease is cancer.

75. Use of a conjugate or mixture according to any one of statements 59to 70, or the pharmaceutical composition according to statement 72 in amethod of medical treatment.

76. A method of medical treatment comprising administering to a patientthe pharmaceutical composition of statement 72.

77. The method of statement 76 wherein the method of medical treatmentis for treating cancer.

78. The method of statement 77, wherein the patient is administered achemotherapeutic agent, in combination with the conjugate.

79. Use of a conjugate or mixture according to any one of statements 59to 70 in a method of manufacture of a medicament for the treatment of aproliferative disease.

80. A method of treating a mammal having a proliferative disease,comprising administering an effective amount of conjugate or mixtureaccording to any one of statements 59 to 70, or the pharmaceuticalcomposition according to statement 72.

81. The compound A:

as a single enantiomer or in an enantiomerically enriched form.

82. A compound with the formula VI:

where Q is as in any one of statements 1 and 3 and 12.

Statements of Invention from 1^(st) Priority Application (P1)

P1-1. A compound with the formula I:

and salts and solvates thereof, wherein R^(L) is a linker for connectionto a cell binding agent, which is selected from:

-   -   (ia):

-   -   wherein    -   Q is:

where Q^(X) is such that Q is an amino-acid residue, a dipeptide residueor a tripeptide residue;

-   -   X is:

-   -   where a=0 to 5, b=0 to 16, c=0 or 1, d=0 to 5;    -   G^(L) is a linker for connecting to a Ligand Unit;    -   (ib):

-   -   where R^(L1) and R^(L2) are independently selected from H and        methyl, or together with the carbon atom to which they are bound        form a cyclopropylene or cyclobutylene group; and    -   e is 0 or 1.

P1-2. The compound according to statement P1-1, wherein R^(L) is offormula Ia.

P1-3. The compound according to statement P1-2, wherein Q is an aminoacid residue.

P1-4. The compound according to statement P1-3, wherein Q is selectedfrom: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp.

P1-5. The compound according to statement P1-2, wherein Q is a dipeptideresidue.

P1-6. The compound according to statement P1-5, wherein Q is selectedfrom:

-   -   ^(NH)-Phe-Lys-^(C═O),    -   ^(NH)-Val-Ala-^(C═O),    -   ^(NH)-Val-Lys-^(C═O),    -   ^(NH)-Ala-Lys-^(C═O),    -   ^(NH)-Val-Cit-^(C═O),    -   ^(NH)-Phe-Cit-^(C═O),    -   ^(NH)-Leu-Cit-^(C═O),    -   ^(NH)-Ile-Cit-^(C═O),    -   ^(NH)-Phe-Arg-^(C═O),    -   ^(NH)-Trp-Cit-^(C═O), and    -   ^(NH)-Gly-Val-^(C═O).

P1-7. The conjugate according to statement P1-6, wherein Q is selectedfrom ^(NH)-Phe-Lys-^(C═O), ^(NH)-Val-Cit-^(C═O) and^(NH)-Val-Ala-^(C═O).

P1-8. The compound according to statement P1-2, wherein Q is atripeptide residue.

P1-9. The compound according to any one of statements P1-2 to P1-8,wherein a is 0 to 3.

P1-10. The compound according to statement P1-9, wherein a is 0 or 1.

P1-11. The compound according to statement P1-9, wherein a is 0.

P1-12. The compound according to any one of statements P1-2 to P1-11,wherein b is 0 to 8.

P1-13. The compound according to statement P1-12, wherein b is 0.

P1-14. The compound according to statement P1-12, wherein b is 4.

P1-15. The compound according to statement P1-12, wherein b is 8.

P1-16. The compound according to any one of statements P1-2 to P1-15,wherein c is 0.

P1-17. The compound according to any one of statements P1-2 to P1-15,wherein c is 1.

P1-18. The compound according to any one of statements P1-2 to P1-17,wherein d is 0 to 3.

P1-19. The compound according to statement P1-18, wherein d is 1 or 2.

P1-20. The compound according to statement P1-19, wherein d is 2.

P1-21. The compound according to any one of statements P1-2 to P1-8,wherein a is 0, c is 1 and d is 2, and b is 0, 4 or 8.

P1-22. The compound according to any one of statements P1-2 to P1-21,wherein G^(L) is selected from

where Ar represents a C₅₋₆ arylene group, and X represents C₁₋₄ alkyl.

P1-23. A compound according to statement P1-22, wherein G^(L) isselected from G^(L1-1) and G^(L1-2).

P1-24. A compound according to statement P1-22, wherein G^(L) isG^(L1-1).

P1-25. The compound according to statement P1-1, wherein R^(L) is offormula Ib.

P1-26. The compound according to statement P1-25, wherein both R^(L1)and R^(L2) are H.

P1-27. The compound according to statement P1-25, wherein R^(L1) is Hand R^(L2) is methyl.

P1-28. The compound according to statement P1-25, wherein both R^(L1)and R^(L2) are methyl.

P1-29. The compound according to statement P1-25, wherein R^(L1) andR^(L2) together with the carbon atom to which they are bound form acyclopropylene group.

P1-30. The compound according to statement P1-25, wherein R^(L1) andR^(L2) together with the carbon atom to which they are bound form acyclobutylene group.

P1-31. The compound according to any one of statements P1-25 to P1-30,wherein e is 0.

P1-32. The compound according to any one of statements P1-25 to P1-30,wherein e is 1.

P1-33. A conjugate of formula IV:

L-(D^(L))_(p)  (IV)

or a pharmaceutically acceptable salt or solvate thereof, wherein L is aLigand unit (i.e., a targeting agent), D^(L) is a Drug Linker unit thatis of formula III:

R^(LL) is a linker connected to the Ligand unit selected from

(ia′):

where Q and X are as defined in any one of statements P1-1 to P1-21 andG^(LL) is a linker connected to a Ligand Unit; and

(ib′):

where R^(L1) and R^(L2) are as defined in any one of statements P1-1 andP1-25 to P1-30; and p is an integer of from 1 to 20.

P1-34. The conjugate according to statement P1-33, wherein G^(LL) isselected from:

where Ar represents a C₅₋₆ arylene group and X represents C₁₋₄ alkyl.

P1-35. The conjugate according to statement P1-34, wherein G^(LL) isselected from G^(LL1-1) and G^(LL1-2).

P1-36. The conjugate according to statement P1-35, wherein G^(LL) isG^(LL1-1).

P1-37. The conjugate according to any one of statements P1-33 to P1-36,wherein the cell binding agent is an antibody or an active fragmentthereof.

P1-38. The conjugate according to statement P1-37, wherein the antibodyor antibody fragment is an antibody or antibody fragment for atumour-associated antigen.

P1-39. The conjugate according to statement P1-38, wherein the antibodyor antibody fragment is an antibody which binds to one or moretumor-associated antigens or cell-surface receptors selected from(1)-(88):

(1) BMPR1B;

(2) E16;

(3) STEAP1;

(4) 0772P;

(5) MPF;

(6) Napi3b;

(7) Sema 5b;

(8) PSCA hlg;

(9) ETBR;

(10) MSG783;

(11) STEAP2;

(12) TrpM4;

(13) CRIPTO;

(14) CD21;

(15) CD79b;

(16) FcRH2;

(17) HER2;

(18) NCA;

(19) MDP;

(20) IL20R-alpha;

(21) Brevican;

(22) EphB2R;

(23) ASLG659;

(24) PSCA;

(25) GEDA;

(26) BAFF-R;

(27) CD22;

(28) CD79a;

(29) CXCR5;

(30) HLA-DOB;

(31) P2X5;

(32) CD72;

(33) LY64;

(34) FcRH1;

(35) IRTA2;

(36) TENB2;

(37) PSMA—FOLH1;

(38) SST;

(38.1) SSTR2;

(38.2) SSTR5;

(38.3) SSTR1;

(38.4) SSTR3;

(38.5) SSTR4;

(39) ITGAV;

(40) ITGB6;

(41) CEACAM5;

(42) MET;

(43) MUC1;

(44) CA9;

(45) EGFRvIII;

(46) CD33;

(47) CD19;

(48) IL2 RA;

(49) AXL;

(50) CD30—TNFRSF8;

(51) BCMA—TNFRSF17;

(52) CT Ags—CTA;

(53) CD174 (Lewis Y)—FUT3;

(54) CLEC14A;

(55) GRP78—HSPA5;

(56) CD70;

(57) Stem Cell specific antigens;

(58) ASG-5;

(59) ENPP3;

(60) PRR4;

(61) GCC—GUCY2C;

(62) Liv-1—SLC39A6;

(63) 5T4;

(64) CD56—NCMA1;

(65) CanAg;

(66) FOLR1;

(67) GPNMB;

(68) TIM-1—HAVCR1;

(69) RG-1/Prostate tumor target Mindin—Mindin/RG-1;

(70) B7-H4—VTCN1;

(71) PTK7;

(72) CD37;

(73) CD138—SDC1;

(74) CD74;

(75) Claudins—CLs;

(76) EGFR;

(77) Her3;

(78) RON—MST1R;

(79) EPHA2;

(80) CD20—MS4A1;

(81) Tenascin C—TNC;

(82) FAP;

(83) DKK-1;

(84) CD52;

(85) CS1—SLAMF7;

(86) Endoglin—ENG;

(87) Annexin A1—ANXA1;

(88) V-CAM (CD106)—VCAM1;

(89) ASCT2 (SLC1A5).

P1-40. The conjugate according to any one of statements P1-37 to P1-39,wherein the antibody or antibody fragment is a cysteine-engineeredantibody.

P1-41. The conjugate according to any one of statements P1-37 to P1-40,wherein the drug loading (p) of drugs (D) to antibody (Ab) is an integerfrom 1 to about 10.

P1-42. The conjugate according to statement P1-41, wherein p is 1, 2, 3,4, 5, 6, 7, 8, 9 or 10.

P1-43. A mixture of conjugates according to any one of statements P1-33to P1-42, wherein the average drug loading per antibody in the mixtureof antibody-drug conjugate compounds is about 1 to about 10.

P1-44. The conjugate or mixture according to any one of statements P1-33to P1-43, for use in therapy.

P1-45. A pharmaceutical composition comprising the conjugate or mixtureof any one of statements P1-33 to P1-43 and a pharmaceuticallyacceptable diluent, carrier or excipient.

P1-46. The conjugate or mixture according to any one of statements P1-33to P1-43, or the pharmaceutical composition according to statementP1-45, for use in the treatment of a proliferative disease in a subject.

P1-47. The conjugate or mixture according to statement P1-46, whereinthe disease is cancer.

P1-48. Use of a conjugate or mixture according to any one of statementsP1-33 to P1-43, or the pharmaceutical composition according to statementP1-45 in a method of medical treatment.

P1-49. A method of medical treatment comprising administering to apatient the pharmaceutical composition of statement P1-45.

P1-50. The method of statement P1-49 wherein the method of medicaltreatment is for treating cancer.

P1-51. The method of statement P1-50, wherein the patient isadministered a chemotherapeutic agent, in combination with theconjugate.

P1-52. Use of a conjugate or mixture according to any one of statementsP1-33 to P1-43 in a method of manufacture of a medicament for thetreatment of a proliferative disease.

P1-53. A method of treating a mammal having a proliferative disease,comprising administering an effective amount of conjugate or mixtureaccording to any one of statements P1-33 to P1-43, or the pharmaceuticalcomposition according to statement P1-45.

P1-54. The compound A:

as a single enantiomer or in an enantiomerically enriched form.

Statements of Invention from 2^(nd) Priority Application (P2)

P2-1. A compound with the formula I:

and salts and solvates thereof, wherein R^(L) is a linker for connectionto a cell binding agent, which is selected from:

-   -   (ia):

-   -   wherein    -   Q is:

where Q^(X) is such that Q is an amino-acid residue, a dipeptideresidue, a tripeptide residue or a tetrapeptide residue;

-   -   X is:

-   -   where a=0 to 5, b1=0 to 16, b2=0 to 16, c=0 or 1, d=0 to 5,        wherein at least b1 or b2=0;    -   G^(L) is a linker for connecting to a Ligand Unit;    -   (ib):

-   -   where R^(L1) and R^(L2) are independently selected from H and        methyl, or together with the carbon atom to which they are bound        form a cyclopropylene or cyclobutylene group; and    -   e is 0 or 1.

P2-2. The compound according to statement P2-1, wherein R^(L) is offormula Ia.

P2-3. The compound according to statement P2-2, wherein Q is an aminoacid residue.

P2-4. The compound according to statement P2-3, wherein Q is selectedfrom: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp.

P2-5. The compound according to statement P2-2, wherein Q is a dipeptideresidue.

P2-6. The compound according to statement P2-5, wherein Q is selectedfrom:

-   -   ^(NH)-Phe-Lys-^(C═O),    -   ^(NH)-Val-Ala-^(C═O),    -   ^(NH)-Val-Lys-^(C═O),    -   ^(NH)-Ala-Lys-^(C═O),    -   ^(NH)-Val-Cit-^(C═O),    -   ^(NH)-Phe-Cit-^(C═O),    -   ^(NH)-Leu-Cit-^(C═O),    -   ^(NH)-Ile-Cit-^(C═O),    -   ^(NH)-Phe-Arg-^(C═O),    -   ^(NH)-Trp-Cit-^(C═O), and    -   ^(NH)-Gly-Val-^(C═O).

P2-7. The compound according to statement P2-6, wherein Q is selectedfrom ^(NH)-Phe-Lys-^(C═O), ^(NH)-Val-Cit-^(C═O) and^(NH)-Val-Ala-^(C═O).

P2-8. The compound according to statement P2-2, wherein Q is atripeptide residue.

P2-9. The compound according to statement P2-8, wherein Q is selectedfrom:

-   -   ^(NH)-Glu-Val-Ala-^(C═O)    -   ^(NH)-Glu-Val-Cit-^(C═O)    -   ^(NH)-αGlu-Val-Ala-^(C═O), and    -   ^(NH)-αGlu-Val-Cit-^(C═O).

P2-10. The compound according to statement P2-2, wherein Q is atetrapeptide residue.

P2-11. The compound according to statement P2-10, wherein Q is selectedfrom:

-   -   ^(NH)-Gly-Gly-Phe-Gly^(C═O); and    -   ^(NH)-Gly-Phe-Gly-Gly^(C═O).

P2-12. The compound according to statement P2-11, wherein Q is:

-   -   ^(NH)-Gly-Gly-Phe-Gly^(C═O).

P2-13. The compound according to any one of statements P2-2 to P2-12,wherein a is 0 to 3.

P2-14. The compound according to statement P2-13, wherein a is 0 or 1.

P2-15. The compound according to statement P2-13, wherein a is 0.

P2-16. The compound according to any one of statements P2-2 to P2-15,wherein b1 is 0 to 8.

P2-17. The compound according to statement P2-16, wherein b1 is 0.

P2-18. The compound according to statement P2-16, wherein b1 is 2.

P2-19. The compound according to statement P2-16, wherein b1 is 3.

P2-20. The compound according to statement P2-16, wherein b1 is 4.

P2-21. The compound according to statement P2-16, wherein b1 is 5.

P2-22. The compound according to statement P2-16, wherein b1 is 8.

P2-23. The compound according to any one of statements P2-2 to P2-15 andP2-17, wherein b2 is 0 to 8.

P2-24. The compound according to statement P2-23, wherein b2 is 0.

P2-25. The compound according to statement P2-23, wherein b2 is 2.

P2-26. The compound according to statement P2-23, wherein b2 is 3.

P2-27. The compound according to statement P2-23, wherein b2 is 4.

P2-28. The compound according to statement P2-23, wherein b2 is 5.

P2-29. The compound according to statement P2-23, wherein b2 is 8.

P2-30. The compound according to any one of statements P2-2 to P2-29,wherein c is 0.

P2-31. The compound according to any one of statements P2-2 to P2-29,wherein c is 1.

P2-32. The compound according to any one of statements P2-2 to P2-31,wherein d is 0 to 3.

P2-33. The compound according to statement P2-32, wherein d is 1 or 2.

P2-34. The compound according to statement P2-32, wherein d is 2.

P2-35. The compound according to any one of statements P2-2 to P2-12,wherein a is 0, b1 is 0, c is 1 and d is 2, and b2 is from 0 to 8.

P2-36. The compound according to statement P2-35, wherein b2 is 0, 2, 3,4, 5 or 8.

P2-37. The compound according to any one of statements P2-2 to P2-12,wherein a is 1, b2 is 0, c is 0 and d is 0, and b1 is from 0 to 8.

P2-38. The compound according to statement P2-37, wherein b1 is 0, 2, 3,4, 5 or 8.

P2-39. The compound according to any one of statements P2-2 to P2-12,wherein a is 0, b1 is 0, c is 0 and d is 1, and b2 is from 0 to 8.

P2-40. The compound according to statement P2-39, wherein b2 is 0, 2, 3,4, 5 or 8.

P2-41. The compound according to any one of statements P2-2 to P2-12,wherein b1 is 0, b2 is 0, c is 0, one of a and d is 0, and the other ofa and d is from 1 to 5.

P2-42. The compound according to statement P2-41, wherein the other of aand d is 1 or 5.

P2-43. The compound according to any one of statements P2-2 to P2-42,wherein G^(L) is selected from

where Ar represents a C₅₋₆ arylene group, and X represents C₁₋₄ alkyl.

P2-44. A compound according to statement P2-43, wherein G^(L) isselected from G^(L1-1) and G^(L1-2).

P2-45. A compound according to statement P2-43, wherein G^(L) isG^(L1-1).

P2-46. The compound according to statement P2-1, wherein R^(L) is offormula Ib.

P2-47. The compound according to statement P2-46, wherein both R^(L1)and R^(L2) are H.

P2-48. The compound according to statement P2-46, wherein R^(L1) is Hand R^(L2) is methyl.

P2-49. The compound according to statement P2-46, wherein both R^(L1)and R^(L2) are methyl.

P2-50. The compound according to statement P2-46, wherein R^(L1) andR^(L2) together with the carbon atom to which they are bound form acyclopropylene group.

P2-51. The compound according to statement P2-46, wherein R^(L1) andR^(L2) together with the carbon atom to which they are bound form acyclobutylene group.

P2-52. The compound according to any one of statements P2-46 to P2-51,wherein e is 0.

P2-53. The compound according to any one of statements P2-46 to P2-51,wherein e is 1.

P2-54. A conjugate of formula IV:

L-(D^(L))_(p)  (IV)

or a pharmaceutically acceptable salt or solvate thereof, wherein L is aLigand unit (i.e., a targeting agent), D^(L) is a Drug Linker unit thatis of formula III:

R^(LL) is a linker connected to the Ligand unit selected from

(ia′):

where Q and X are as defined in any one of statements P2-1 to P2-42 andG^(LL) is a linker connected to a Ligand Unit; and

(ib′):

where R^(L1) and R^(L2) are as defined in any one of statements P2-1 andP2-47 to P2-51; and

p is an integer of from 1 to 20.

P2-55. The conjugate according to statement P2-54, wherein G^(LL) isselected from:

where Ar represents a C₅₋₆ arylene group and X represents C₁₋₄ alkyl.

P2-56. The conjugate according to statement P2-55, wherein G^(LL) isselected from G^(LL1-1) and G^(LL1-2).

P2-57. The conjugate according to statement P2-56, wherein G^(LL) isG^(LL1-1).

P2-58. The conjugate according to any one of statements P2-54 to P2-57,wherein the Ligand Unit is an antibody or an active fragment thereof.

P2-59. The conjugate according to statement P2-58, wherein the antibodyor antibody fragment is an antibody or antibody fragment for atumour-associated antigen.

P2-60. The conjugate according to statement P2-59, wherein the antibodyor antibody fragment is an antibody which binds to one or moretumor-associated antigens or cell-surface receptors selected from(1)-(89):

(1) BMPR1B;

(2) E16;

(3) STEAP1;

(4) 0772P;

(5) MPF;

(6) Napi3b;

(7) Sema 5b;

(8) PSCA hlg;

(9) ETBR;

(10) MSG783;

(11) STEAP2;

(12) TrpM4;

(13) CRIPTO;

(14) CD21;

(15) CD79b;

(16) FcRH2;

(17) HER2;

(18) NCA;

(19) MDP;

(20) IL20R-alpha;

(21) Brevican;

(22) EphB2R;

(23) ASLG659;

(24) PSCA;

(25) GEDA;

(26) BAFF-R;

(27) CD22;

(28) CD79a;

(29) CXCR5;

(30) HLA-DOB;

(31) P2X5;

(32) CD72;

(33) LY64;

(34) FcRH1;

(35) IRTA2;

(36) TENB2;

(37) PSMA—FOLH1;

(38) SST;

(38.1) SSTR2;

(38.2) SSTR5;

(38.3) SSTR1;

(38.4) SSTR3;

(38.5) SSTR4;

(39) ITGAV;

(40) ITGB6;

(41) CEACAM5;

(42) MET;

(43) MUC1;

(44) CA9;

(45) EGFRvIII;

(46) CD33;

(47) CD19;

(48) IL2RA;

(49) AXL;

(50) CD30—TNFRSF8;

(51) BCMA—TNFRSF17;

(52) CT Ags—CTA;

(53) CD174 (Lewis Y)—FUT3;

(54) CLEC14A;

(55) GRP78—HSPA5;

(56) CD70;

(57) Stem Cell specific antigens;

(58) ASG-5;

(59) ENPP3;

(60) PRR4;

(61) GCC—GUCY2C;

(62) Liv-1—SLC39A6;

(63) 5T4;

(64) CD56—NCMA1;

(65) CanAg;

(66) FOLR1;

(67) GPNMB;

(68) TIM-1—HAVCR1;

(69) RG-1/Prostate tumor target Mindin—Mindin/RG-1;

(70) B7-H4—VTCN1;

(71) PTK7;

(72) CD37;

(73) CD138—SDC1;

(74) CD74;

(75) Claudins—CLs;

(76) EGFR;

(77) Her3;

(78) RON—MST1R;

(79) EPHA2;

(80) CD20—MS4A1;

(81) Tenascin C—TNC;

(82) FAP;

(83) DKK-1;

(84) CD52;

(85) CS1—SLAMF7;

(86) Endoglin—ENG;

(87) Annexin A1—ANXA1;

(88) V-CAM (CD106)—VCAM1;

(89) ASCT2 (SLC1A5).

P2-61. The conjugate according to any one of statements P2-58 to P2-60,wherein the antibody or antibody fragment is a cysteine-engineeredantibody.

P2-62. The conjugate according to any one of statements P2-58 to P2-61,wherein the drug loading (p) of drugs (D) to antibody (Ab) is an integerfrom 1 to about 10.

P2-63. The conjugate according to statement P2-62, wherein p is 1, 2, 3,4, 5, 6, 7, 8, 9 or 10.

P2-64. A mixture of conjugates according to any one of statements P2-58to P2-63, wherein the average drug loading per antibody in the mixtureof antibody-drug conjugates is about 1 to about 10.

P2-65. The conjugate or mixture according to any one of statements P2-54to P2-64, for use in therapy.

P2-66. A pharmaceutical composition comprising the conjugate or mixtureof any one of statements P2-54 to P2-64 and a pharmaceuticallyacceptable diluent, carrier or excipient.

P2-67. The conjugate or mixture according to any one of statements P2-54to P2-64, or the pharmaceutical composition according to statementP2-66, for use in the treatment of a proliferative disease in a subject.

P2-68. The conjugate, mixture or pharmaceutical composition according tostatement P2-67, wherein the disease is cancer.

P2-69. Use of a conjugate or mixture according to any one of statementsP2-54 to P2-64, or the pharmaceutical composition according to statementP2-66 in a method of medical treatment.

P2-70. A method of medical treatment comprising administering to apatient the pharmaceutical composition of statement P2-66.

P2-71. The method of statement P2-70 wherein the method of medicaltreatment is for treating cancer.

P2-72. The method of statement P2-71, wherein the patient isadministered a chemotherapeutic agent, in combination with theconjugate.

P2-73. Use of a conjugate or mixture according to any one of statementsP2-54 to P2-64 in a method of manufacture of a medicament for thetreatment of a proliferative disease.

P2-74. A method of treating a mammal having a proliferative disease,comprising administering an effective amount of conjugate or mixtureaccording to any one of statements P2-54 to P2-64, or the pharmaceuticalcomposition according to statement P2-66.

P2-75. The compound A:

as a single enantiomer or in an enantiomerically enriched form.

1. A compound with the formula I:

and salts and solvates thereof, wherein R^(L) is a linker for connection to a Ligand Unit, which is selected from: (ia):

wherein Q is:

where Q^(X) is such that Q is an amino-acid residue, a dipeptide residue, a tripeptide residue or a tetrapeptide residue; X is:

where a=0 to 5, b1=0 to 16, b2=0 to 16, c1=0 or 1, c2=0 or 1, d=0 to 5, wherein at least b1 or b2=0 and at least c1 or c2=0; G^(L) is a linker for connecting to a Ligand Unit; (ib):

where R^(L1) and R^(L2) are independently selected from H and methyl, or together with the carbon atom to which they are bound form a cyclopropylene or cyclobutylene group; and e is 0 or
 1. 2. The compound according to claim 1, wherein R^(L) is of formula Ia.
 3. The compound according to claim 2, wherein Q is: (a) an amino acid residue selected from: Phe, Lys, Val, Ala, Cit, Leu, Ile, Arg, and Trp; or (b) a dipeptide residue selected from: ^(NH)-Phe-Lys-^(C═O), ^(NH)-Val-Ala-^(C═O), ^(NH)-Val-Lys-^(C═O), ^(NH) Ala-Lys-^(C═O), ^(NH)-Val-Cit-^(C═O), ^(NH)-Phe-Cit-^(C═O), ^(NH)-Leu-Cit-^(C═O), ^(NH)-Ile-Cit-^(C═O), ^(NH)-Phe-Arg-^(C═O), ^(NH)-Trp-Cit-^(C═O), and ^(NH)-Gly-Val-^(C═O); or (c) a tripeptide residue selected from: ^(NH)-Glu-Val-Ala-^(C═O), ^(NH)-Glu-Val-Cit-^(C═O), ^(NH)-αGlu-Val-Ala-^(C═O), and ^(NH)-αGlu-Val-Cit-^(C═O); or (d) a tetrapeptide residue selected from: ^(NH)-Gly-Gly-Phe-Gly^(C═O); and ^(NH)-Gly-Phe-Gly-Gly^(C═O).
 4. The compound according to either claim 2 or claim 3, wherein a is: (a) 0 to 3; or (b) 0 or 1; or (c)
 0. 5. The compound according to any one of claims 2 to 4, wherein b1 is: (a) 0 to 8; or (b) 0; or (c) 2; or (d) 3; or (e) 4; or (f) 5; or (g)
 8. 6. The compound according to any one of claims 2 to 4, wherein b2 is: (a) 0 to 8; or (b) 0; or (c) 2; or (d) 3; or (e) 4; or (f) 5; or (g)
 8. 7. The compound according any to one of claims 2 to 6, wherein (i) c1 is: (a) 0; or (b) 1; and (ii) c2 is: (a) 0; or (b) 1; wherein at least one of c1 and c2 is
 0. 8. The compound according to any one of claims 2 to 7, wherein d is: (a) 0 to 3; or (b) 1 or 2; or (c) 2; or (d)
 5. 9. The compound according to any one of claims 2 to 8, wherein: (a) a is 0, b1 is 0, c1 is 1, c2 is 0 and d is 2, and b2 is 0, 2, 3, 4, 5 or 8; or (b) a is 1, b2 is 0, c1 is 0, c2 is 0 and d is 0, and b1 is 0, 2, 3, 4, 5 or 8; or (c) a is 0, b1 is 0, c1 is 0, c2 is 0 and d is 1, and b2 is 0, 2, 3, 4, 5 or 8; or (d) b1 is 0, b2 is 0, c1 is 0, c2 is 0, one of a and d is 0, and the other of a and d is 1 or 5; or (e) a is 1, b2 is 0, c1 is 0, c2 is 1, d is 2, and b1 is 0, 2, 3, 4, 5 or
 8. 10. The compound according to any one of claims 2 to 9, wherein G^(L) is selected from

where Ar represents a C₅₋₆ arylene group, and X represents C₁₋₄ alkyl.
 11. A compound according to claim 10, wherein G^(L) is selected from G^(L1-1) and G^(L1-2).
 12. The compound according to claim 1, wherein R^(L) is of formula Ib, and: (a) both R^(L1) and R^(L2) are H; or (b) R^(L1) is H and R^(L2) is methyl; or (c) both R^(L1) and R^(L2) are methyl; or (d) wherein R^(L1) and R^(L2) together with the carbon atom to which they are bound form a cyclopropylene group; or (e) wherein R^(L1) and R^(L2) together with the carbon atom to which they are bound form a cyclobutylene group.
 13. A conjugate of formula IV: L-(D^(L))_(p)  (IV) or a pharmaceutically acceptable salt or solvate thereof, wherein L is a Ligand unit, D^(L) is a Drug Linker unit that is of formula III:

R^(LL) is a linker connected to the Ligand unit selected from (ia′):

where Q and X are as defined in any one of claims 1 to 9 and G^(LL) is a linker connected to a Ligand Unit; and (ib′):

where R^(L1) and R^(L2) are as defined in either claim 1 or claim 12; and p is an integer of from 1 to
 20. 14. The conjugate according to claim 13, wherein G^(LL) is selected from:

where Ar represents a C₅₋₆ arylene group and X represents C₁₋₄ alkyl.
 15. The conjugate according to claim 14, wherein G^(LL) is selected from G^(LL1-1) and G^(LL1-2).
 16. The conjugate according to any one of claims 13 to 15, wherein the Ligand Unit is an antibody or an active fragment thereof.
 17. The conjugate according to claim 16, wherein the drug loading (p) of drugs (D) to antibody (Ab) is an integer from 1 to about
 10. 18. A mixture of conjugates according to either claim 16 or claim 17, wherein the average drug loading per antibody in the mixture of antibody-drug conjugates is about 1 to about
 10. 19. A pharmaceutical composition comprising the conjugate or mixture of any one of claims 13 to 18 and a pharmaceutically acceptable diluent, carrier or excipient.
 20. The conjugate or mixture according to any one of claims 13 to 18, or the pharmaceutical composition according to claim 19, for use in the treatment of a proliferative disease in a subject.
 21. The conjugate, mixture or pharmaceutical composition according to claim 20, wherein the disease is cancer.
 22. Use of a conjugate or mixture according to any one of claims 13 to 18, or the pharmaceutical composition according to claim 19 in a method of medical treatment.
 23. A method of medical treatment comprising administering to a patient the pharmaceutical composition of claim
 19. 24. The method of claim 23 wherein the method of medical treatment is for treating cancer.
 25. The compound A:

as a single enantiomer or in an enantiomerically enriched form.
 26. A compound with the formula VI:

where Q is as in either claim 1 or
 3. 